JP2017537090A - Combination therapy comprising OX40 binding agonist and PD-1 axis binding antagonist - Google Patents

Abstract

The present invention provides compositions and methods for treating cancer. This includes administering a PD-1 axis binding antagonist and an OX40 binding agonist. [Selection] Figure 8A

Description

This application claims the benefit of priority of US Provisional Application No. 62 / 080,991 filed November 17, 2014 and 62 / 093,400 filed December 17, 2014. Each of which is incorporated herein by reference in its entirety.

Sequence Listing The contents of the following submissions in ASCII text files are hereby incorporated by reference in their entirety: Sequence Listing in computer readable form (CRF) (file name: 14463926640640 SeqList.txt, date recorded: November 12, 2015 Day, size: 73KB).

  The present invention relates to a method for treating cancer by administering a PD-1 axis binding antagonist and an OX40 binding agonist.

  Supplying two distinct signals to T cells is a widely accepted model for lymphocyte activation of resting T lymphocytes by antigen presenting cells (APC). Lufferty et al, Aust. J. et al. Exp. Biol. Med. Sci 53: 27-42 (1975). This model further provides a distinction between self-tolerance and non-self tolerance and immune tolerance. Bretscher et al, Science 169: 1042-1049 (1970), Bretscher, P. et al. A. , Proc. Nat. Acad. Sci. USA 96: 185-190 (1999), Jenkins et al, J. MoI. Exp. Med. 165: 302-319 (1987). Primary signals, ie antigen-specific signals, are transmitted through the T cell receptor (TCR) after recognition of foreign antigen peptides presented in the context of the major histocompatibility complex (MHC). The second signal, the costimulatory signal, is delivered to the T cell by a costimulatory molecule expressed in the antigen presenting cell (APC), which promotes clonal proliferation, cytokine secretion, and effector function. Lenschow et al. , Ann. Rev. Immunol. 14: 233 (1996). In the absence of costimulation, T cells can become refractory to antigen stimulation, do not initiate an effective immune response, and can further lead to exhaustion or tolerance to foreign antigens.

  In this two-signal model, T cells receive both positive and negative secondary costimulatory signals. Control of such positive and negative signals is crucial to maintaining immune tolerance and preventing autoimmunity while maximizing the host's protective immune response. While a negative secondary signal appears to be necessary for induction of T cell tolerance, a positive signal promotes T cell activation. Although this simple two-signal model still provides a legitimate explanation for naive lymphocytes, the host immune response is a dynamic process, and costimulatory signals can also be supplied to antigen-exposed T cells. The mechanism of costimulation is of therapeutic interest because it has been shown that manipulation of the costimulatory signal provides a means to either enhance or terminate the cell-based immune response. Recently, it has been discovered that T cell dysfunction or anergy coincides with induced sustained expression of the inhibitory receptor programmed death 1 polypeptide (PD-1). As a result, therapeutic targets for PD-1 and other molecules that signal by interaction with PD-1, such as programmed death ligand 1 (PD-Ll) and programmed death ligand 2 (PD-L2), are extremely This is an interesting field.

  PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Internal. Immun. 2007 19 (7): 813) (Thompson RH et al., Cancer Res 2006, 66 (7): 3381). Interestingly, the majority of tumor infiltrating T lymphocytes predominantly express PD-1 as opposed to T lymphocytes in normal tissues and peripheral blood T lymphocytes, and PD- in tumor reactive T cells. We show that an upregulation of 1 can contribute to impaired anti-tumor immune response (Blood 2009 114 (8): 1537). This may be due to the utilization of PD-L1 signaling mediated by PD-L1-expressing tumor cells interacting with PD-1-expressing T cells, resulting in attenuation of T cell activation and avoidance of immune surveillance (Sharp et al., Nat Rev 2002) (Keir ME et al., 2008 Annu. Rev. Immunol. 26: 677). Thus, inhibition of PD-L1 / PD-1 interaction can enhance CD8 + T cell-mediated tumor death.

  The therapeutic target PD-1, and other molecules that signal by interaction with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) are very interesting areas. . Inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity for the treatment of cancer (eg, tumor immunity) and infections, eg, both acute and chronic (eg, persistent) infections. ing. Optimal therapeutic treatment can combine blocking PD-1 receptor / ligand interaction with agents that directly inhibit tumor growth. There remains a need for optimal therapies to treat, stabilize, prevent and / or delay the onset of various cancers.

  The mechanism of costimulation is of therapeutic interest because it has been shown that manipulation of the costimulatory signal provides a means to either enhance or terminate the cell-based immune response. OX40 (also known as CD34, TNFRSF4, or ACT35 antigen), a member of the tumor necrosis factor receptor superfamily, supplies costimulatory signals to CD4 + and CD8 + T cells and enhances cell proliferation, survival, effector function, and migration. obtain. OX40 signaling also enhances memory T cell development and function. OX40 is not constitutively expressed on naive T cells, but is induced after involvement of the T cell receptor (TCR). OX40L, which is a ligand for OX40, is mainly expressed in antigen-presenting cells. OX40 is highly expressed by activated CD4 + T cells, activated CD8 + T cells, memory T cells, and regulatory T (Treg) cells.

  By combining OX40 signaling with other deregulated signaling pathways in tumor cells, therapeutic efficacy can be further enhanced. Accordingly, there remains a need for optimal therapies to treat or delay the onset of various cancers, immune related diseases, and T cell dysfunction disorders.

  All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are specifically and individually indicated so that each individual reference is incorporated by reference. As if, they are incorporated herein by reference in their entirety.

  In one aspect, a method of treating cancer or delaying its progression in an individual comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody). Provided in the description.

  In another aspect, a method of treating cancer or delaying its progression in an individual comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist, wherein the individual has cancer Alternatively, provided herein is a method wherein cells in a tumor sample of cancer from an individual who have been diagnosed with cancer do not express PD-L1.

  In another aspect, a method of treating cancer or delaying its progression in an individual comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist, wherein the individual has cancer Or a method in which a cell in a tumor sample of cancer from an individual expresses PD-L1 has been diagnosed with cancer.

  In another aspect, provided herein is a method of enhancing immune function in an individual with cancer comprising administering an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody). Is done.

  In another aspect, a method of enhancing immune function in an individual having cancer, comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist, wherein the individual has been diagnosed with cancer Provided herein are methods, wherein cells in a tumor sample of cancer from an individual do not express PD-L1.

  In another aspect, a method of enhancing immune function in an individual having cancer, comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist, wherein the individual has been diagnosed with cancer Provided herein are methods, wherein cells in a tumor sample of cancer from an individual express PD-L1.

  In a further aspect, provided herein is a method of treating an infection (eg, a bacterial or viral infection or other pathogen infection). In some embodiments, the infection is a viral infection and / or a bacterial infection. In some embodiments, the infection is a pathogen infection. In some embodiments, the infection is an acute infection. In some embodiments, the infection is a chronic infection.

  In another aspect, the use of a human PD-1 axis binding antagonist in the manufacture of a medicament for treating cancer or delaying its progression in an individual (or treating infection in some embodiments). Wherein the medicament comprises a human PD-1 axis binding antagonist and any pharmaceutically acceptable carrier, and the treatment comprises a medicament, an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) and any pharmaceutically acceptable Use is provided herein, including administration in combination with a composition comprising a carrier to be prepared.

  In another aspect, an OX40 binding agonist (eg, an anti-human OX40 agonist antibody in the manufacture of a medicament for treating cancer or delaying its progression in an individual (or treating infection in some embodiments) in an individual. ), Wherein the medicament comprises an OX40 binding agonist and any pharmaceutically acceptable carrier, and the treatment comprises a medicament, a human PD-1 axis binding antagonist and any pharmaceutically acceptable carrier. Use is provided herein, including administration in combination with a composition comprising.

  In another aspect, a human PD-1 axis binding antagonist and any pharmaceutically acceptable carrier for use in treating cancer or delaying its progression in an individual (or in some embodiments, treating infection). Wherein the treatment comprises administration of said composition in combination with a second composition, wherein the second composition comprises an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) and any Provided herein are compositions comprising a pharmaceutically acceptable carrier.

  In another aspect, an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) and any pharmaceutical for use in treating cancer or delaying its progression in an individual (or treatment of infection in some embodiments) A composition comprising an acceptable carrier, wherein the treatment comprises administration of the composition in combination with a second composition, wherein the second composition comprises a human PD-1 axis binding antagonist and any pharmaceutical agent. Compositions comprising an pharmaceutically acceptable carrier are provided herein.

  In another aspect, a medicament comprising a PD-1 axis binding antagonist and any pharmaceutically acceptable carrier and a package insert treating or delaying progression of cancer in an individual (or some In an embodiment of the present invention, instructions for administration of the agent in combination with a composition comprising an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) and any pharmaceutically acceptable carrier for treating an infection) A kit containing the package insert is provided herein.

  In another aspect, a first medicament comprising a PD-1 axis binding antagonist and any pharmaceutically acceptable carrier, an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) and any pharmaceutically acceptable A kit comprising a second medicament comprising a carrier is provided herein. In some embodiments, the kit is a first medicament and a second medicament for treating cancer or delaying its progression in an individual (or treating infection in some embodiments). A package insert containing instructions for administration of

  In another aspect, a medicament comprising an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) and any pharmaceutically acceptable carrier, and a package insert, wherein the cancer is treated or progressed in an individual. Instructions for co-administration of the medicament with a composition comprising a PD-1 axis binding antagonist and any pharmaceutically acceptable carrier for delaying (or in some embodiments treating infection) A kit containing the package insert is provided herein.

  In some embodiments, the cancer is breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma Neuroblastoma, or hepatocellular carcinoma.

  In some embodiments, the individual has cancer or has been diagnosed with cancer.

  In some embodiments, the cancer cells (in a sample of cancer from the individual) do not express PD-L1. In some embodiments, the PD-L1 biomarker is not present in the sample when included in 0% of the sample. In some embodiments, PD-L1 biomarker expression is determined by protein expression (eg, by immunohistochemistry (IHC) methods).

  In some embodiments, the cancer cells (derived from a sample of cancer from an individual) express PD-L1. In some embodiments, a PD-L1 biomarker is present in a sample when included in more than 0% of the sample. In some embodiments, the PD-L1 biomarker is detected in the sample by protein expression. In some embodiments, protein expression is determined by immunohistochemistry (IHC). In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody. In some embodiments, the PD-L1 biomarker is detected as a weak staining intensity by IHC, a moderate staining intensity by IHC, or a strong staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody, and the PD-L1 biomarker is detected as moderate staining intensity by IHC, by IHC or as strong staining intensity. Is done.

  In some embodiments, the individual has a cancer that is resistant to a PD-1 axis binding antagonist. In some embodiments, the individual is refractory to a PD-1 axis binding antagonist. In some embodiments, the patient did not show an effective response to the PD-1 axis binding antagonist.

  In some embodiments, the individual has a cancer that has a high amount of T cell infiltrates (eg, determined using a diagnostic test). In some embodiments, the individual has a cancer that has a small amount or an essentially undetectable T cell infiltrate (eg, determined using a diagnostic test).

  In some embodiments of the methods, uses, compositions, and kits described above and herein, treatment with human PD-1 axis binding antagonists and OX40 binding agonists (eg, anti-human OX40 agonist antibodies) or they Administration results in a sustained response in the individual after cessation of treatment.

  In some embodiments, the combination treatment of an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) and a PD-1 axis binding antagonist (eg, an anti-PD-1 or anti-PDL1 antibody) is synergistic, thereby The effective dose of the OX40 binding agent (eg, anti-human OX40 agonist antibody) in combination is reduced compared to the effective dose of the OX40 binding agent (eg, anti-human OX40 agonist antibody) as a single agent.

  In some embodiments, the OX40 binding agonist is administered before the PD-1 axis binding antagonist, simultaneously with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist and the OX40 binding agonist are present in the same composition.

  In some embodiments, the PD-1 axis binding antagonist and the OX40 binding agonist are present in separate compositions. In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PDL1 binding antagonist, and a PDL2 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PDL1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PDL2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PDL1 and PDL2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is nivolumab. In some embodiments, the PD-1 binding antagonist is pembrolizumab. In some embodiments, the PD-1 binding antagonist is CT-011. In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-1 axis binding antagonist is a PDL1 binding antagonist. In some embodiments, the PDL1 binding antagonist inhibits binding of PDL1 to PD-1. In some embodiments, the PDL1 binding antagonist inhibits PDL1 binding to B7-1. In some embodiments, the PDL1 binding antagonist inhibits binding of PDL1 to both PD-1 and B7-1. In some embodiments, the PDL1 binding antagonist is an anti-PDL1 antibody. In some embodiments, the anti-PDL1 antibody is a monoclonal antibody. In some embodiments, the anti-PDL1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab ') 2 fragments. In some embodiments, the anti-PDL1 antibody is a humanized antibody or a human antibody. In some embodiments, the PDL1 binding antagonist is YW243.55. Selected from the group consisting of S70, MPDL3280A, MDX-1105, and MEDI4736. In some embodiments, the antibody comprises a heavy chain comprising the HVR-H1 sequence GFTFSDSWIH (SEQ ID NO: 1), the HVR-H2 sequence AWISPYGGSTYYADSVGK (SEQ ID NO: 2), and the HVR-H3 sequence RHWPGGFDY (SEQ ID NO: 3); And a light chain comprising the HVR-L1 sequence RASQDVSTAVA (SEQ ID NO: 4), the HVR-L2 sequence SASFLYS (SEQ ID NO: 5), and the HVR-L3 sequence QQYLYHPAT (SEQ ID NO: 6). In some embodiments, the antibody comprises the amino acid sequence IbuikyuerubuiiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesudiesudaburyuaieichidaburyubuiarukyueiPijikeijieruidaburyubuieidaburyuaiesuPiwaijijiesutiwaiwaieidiesubuikeijiaruefutiaiesueiditiesukeienutieiwaierukyuemuenuesueruarueiiditieibuiYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7) or a heavy chain variable region comprising the EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 8), amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWY QKPGKAPKLLIY including SASF EruwaiesujibuiPiesuaruefuesujiesujiesujitidiefutierutiaiesuesuerukyuPiidiefuATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9) and a light chain variable region. In some embodiments, the PD-1 axis binding antagonist is a PDL2 binding antagonist. In some embodiments, the PDL2 binding antagonist is an antibody. In some embodiments, the PDL2 binding antagonist is an immunoadhesin. In some embodiments, the PD-1 axis binding antagonist is an antibody (eg, an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-PDL2 antibody) that includes one or more aglycosylation site mutations (eg, substitutions). . In some embodiments, the substitution mutation comprises one or more substitutions at amino acid positions N297, L234, L235, and D265 (EU numbering). In some embodiments, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, and D265A (EU numbering). In some embodiments, the antibody is human IgG1.

  In some embodiments, the OX40 binding agonist is selected from the group consisting of an OX40 agonist antibody, an OX40L agonist fragment, an OX40 oligomer receptor, and an OX40 immunoadhesin. In some embodiments, the OX40 agonist antibody binds to human OX40. In some embodiments, the OX40 agonist antibody is any of the anti-human OX40 agonist antibodies disclosed herein (eg, paragraphs 198-226). In some embodiments, the OX40 agonist antibody is MEDI6469, MEDI0562, or MEDI6383. In some embodiments, the OX40 agonist antibody is a full-length IgG1 antibody. In some embodiments, the OX40 binding agonist is a trimeric OX40L-Fc protein. In some embodiments, the OX40 binding agonist is a trimeric OX40L fusion protein as described in US Pat. No. 7,959,925. In some embodiments, the OX40 binding agonist comprises one or more extracellular domains of OX40L. In some embodiments that may be combined with any other embodiment, the OX40 binding agonist (eg, OX40 agonist antibody) is not MEDI6383. In some embodiments that can be combined with any other embodiment, the OX40 binding agonist (eg, OX40 agonist antibody) is not MEDI0562. In some embodiments, the OX40 binding agonist (eg, OX40 agonist antibody) is a human antibody and / or a humanized antibody. In some embodiments, the OX40 binding agonist (eg, OX40 agonist antibody) is a depleted anti-human OX40 antibody (eg, depletes cells that express human OX40). In some embodiments, the human OX40 expressing cells are CD4 + effector T cells. In some embodiments, the human OX40 expressing cell is a Treg cell. In some embodiments, the depletion is due to ADCC and / or phagocytosis. In some embodiments, the antibody mediates ADCC by binding FcγR expressed by human effector cells and activating human effector cell function. In some embodiments, the antibody mediates phagocytosis by binding FcγR expressed by human effector cells and activating human effector cell function. In some embodiments, the human effector cells are selected from macrophages, natural killer (NK) cells, monocytes, and neutrophils. In some embodiments, the human effector cell is a macrophage. In some embodiments, the OX40 binding agonist (eg, OX40 agonist antibody) has a functional Fc region. In some embodiments, the effector function of the functional Fc region is ADCC. In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region is human IgG4.

  In some embodiments of the methods, uses, compositions, and kits described above and herein, a PD-1 axis binding antagonist and / or OX40 binding agonist (eg, an anti-human OX40 agonist antibody) is administered intravenously. Administered intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments of the methods, uses, compositions, and kits described above and herein, the treatment comprises administering a chemotherapeutic agent to treat or delay the progression of cancer in the individual. In addition. In some embodiments, the individual has been treated with a chemotherapeutic agent prior to the combination treatment of a PD-1 axis binding antagonist and an OX40 binding agonist. In some embodiments, an individual treated with a combination of a PD-1 axis binding antagonist and / or an OX40 binding agonist is refractory to chemotherapeutic treatment. Some embodiments of the methods, uses, compositions and kits described throughout this application further comprise administering a chemotherapeutic agent to treat or delay the progression of cancer.

  In some embodiments of the methods, uses, compositions, and kits described above and herein, CD8 T cells in an individual have enhanced priming, activation, compared to prior to administration of the combination. Has proliferation and / or cytolytic activity. In some embodiments, the number of CD8 T cells is increased compared to before administration of the combination. In some embodiments, the CD8 T cell is an antigen-specific CD8 T cell. In some embodiments, Treg function is suppressed as compared to prior to administration of the combination. In some embodiments, T cell depletion is reduced compared to before administration of the combination. In some embodiments, the number of Treg cells is reduced compared to before administration of the combination. In some embodiments, plasma interferon gamma is increased compared to before administration of the combination. In some embodiments, the number of memory effector T cells is increased compared to before administration of the combination. In some embodiments, memory effector T cell activation and / or proliferation is increased compared to prior to administration of the combination. In some embodiments, memory effector T cells are detected in peripheral blood. In some embodiments, the detection of memory effector T cells is by detection of CXCR3.

  Expression level of one or more marker gene (s), protein (s) (eg, cytokines, eg, gamma interferon), and / or cellular composition in a sample (eg, peripheral blood) containing leukocytes obtained from a subject For example, by measuring the percentage of Tregs and / or the absolute number of Tregs, eg, the number of CD8 + effector T cells (subject is a PD-1 axis binding antagonist and OX40 binding agonist (eg anti-human OX40 agonist antibody)) Treated with one or more marker genes selected from a T cell marker gene or a memory T cell marker gene (eg, a marker for effector memory T cells) and the treatment relative to a reference , One or more marker genes, proteins (compounds) in a sample obtained from the subject And / or determining that it exhibits pharmacodynamic activity based on the expression level of the cell composition (increased expression level of one or more marker genes compared to the reference is a drug for OX40 agonist treatment) Provided herein is a method of monitoring the pharmacodynamic activity of an OX40 agonist treatment. The expression level of the marker gene, protein, and / or cell composition is measured by one or more methods described herein. In some embodiments, measuring proliferating CD8 + T cell levels (eg, percentage of Ki67 + / total CD8 + T cells) in a sample (eg, peripheral blood sample) from an individual (reference (eg, level prior to combination treatment)). A method for monitoring the pharmacodynamic activity of a combination treatment of an OX40 agonist treatment and a PD-1 axis binding antagonist, comprising an increase in proliferating CD8 + T cell levels in the sample compared to Provided herein. In some embodiments, measuring activated CD8 + T cell levels (eg, the percentage of CXCR3 + / total CD8 + T cells) in a sample from an individual (eg, a peripheral blood sample) (eg, baseline (eg, level prior to combination treatment). Monitoring the pharmacodynamic activity of the combination treatment of OX40 agonist treatment and PD-1 axis binding antagonist, including an increase in activated CD8 + T cell levels in the sample compared to A method is provided herein.

  Expression level of one or more marker gene (s), protein (s) (eg, cytokines, eg, gamma interferon), and / or cellular composition in a sample (eg, peripheral blood) in white blood cells obtained from a subject For example, measuring the percentage of Tregs and / or the absolute number of Tregs, eg, the number of CD8 + effector T cells in a peripheral blood sample (the subject is a PD-1 axis binding antagonist and OX40 binding agonist (eg, anti-human One or more marker genes selected from a T cell marker gene or a memory T cell marker gene (eg, an effector memory T cell marker) and a subject As compared to one or more marker genes, tampers in a sample obtained from a subject. Classifying as responsive or non-responsive to this treatment based on quality (s) and / or expression level of the cellular composition (increased expression level of one or more marker genes compared to the reference) Provided herein is a method of monitoring a subject's responsiveness to OX40 agonist treatment). The expression level of the marker gene, protein, and / or cell composition is measured by one or more methods described herein. In some embodiments, measuring proliferating CD8 + T cell levels (eg, percentage of Ki67 + / total CD8 + T cells) in a sample (eg, peripheral blood sample) from an individual (reference (eg, level prior to combination treatment)). A method of monitoring the responsiveness of a combination treatment of an OX40 agonist treatment and a PD-1 axis binding antagonist, including an increase in proliferating CD8 + T cell levels in the sample compared to Provided to. In some embodiments, measuring activated CD8 + T cell levels (eg, the percentage of CXCR3 + / total CD8 + T cells) in a sample from an individual (eg, a peripheral blood sample) (eg, baseline (eg, level prior to combination treatment). The present invention relates to a method for monitoring the responsiveness of a combination treatment of an OX40 agonist treatment and a PD-1 axis binding antagonist, comprising an increase in the level of activated CD8 + T cells in the sample compared to Provided in the description.

  It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further illustrated by the following detailed description.

Tumor infiltrating CD8 + T cells express high levels of PD-1 inhibitory receptors in the CT26 colorectal syngeneic tumor model (control treated mice). Approximately half of PD-1 expressing CD8 + TIL also express OX40. Representative flow cytometry dot plot of 1 of 5 mice 2 days after initiation of treatment with control antibody. (FIG. 2A) Treatment with anti-OX40 agonist antibody alone and combined treatment with anti-OX40 agonist antibody and anti-PDL1 antagonist antibody significantly reduced the proportion of intratumoral Foxp3 + regulatory T cells (relative to the total number of CD45 + cells). For both (FIG. 2A) and (FIG. 2B), the data are from 9 days after the start of treatment and each symbol represents an individual mouse. Mice were dosed BIW (twice a week) with control antibody or anti-PDL1 antibody on day 1 at the initial dose of 10 mg / kg (intravenous) followed by 5 mg / kg (intraperitoneal). Anti-OX40 agonist antibody was dosed on day 1 at the initial dose of 0.1 mg / kg (intravenous) followed by 0.1 mg / kg (intraperitoneal) TIW (3 times per week). (FIG. 2B) Treatment with anti-OX40 agonist antibody alone and combined treatment with anti-OX40 agonist antibody and anti-PDL1 antagonist antibody significantly reduced the absolute number of intratumoral Foxp3 + regulatory T cells in the CT26 colorectal tumor model. For both (FIG. 2A) and (FIG. 2B), the data are from 9 days after the start of treatment and each symbol represents an individual mouse. Mice were dosed BIW (twice a week) with control antibody or anti-PDL1 antibody on day 1 at the initial dose of 10 mg / kg (intravenous) followed by 5 mg / kg (intraperitoneal). Anti-OX40 agonist antibody was dosed on day 1 at the initial dose of 0.1 mg / kg (intravenous) followed by 0.1 mg / kg (intraperitoneal) TIW (3 times per week). Treatment with anti-OX40 agonist antibody increased PDL1 expression in intratumoral myeloid (CD11b + Gr-1 low / intermediate) cells and (FIG. 3B) tumor cells in the CT26 colorectal syngeneic tumor model. Data are from 9 days after the start of treatment. Each dot / square represents one individual mouse. PDL1 expression measured by geometric mean fluorescence intensity (geometric MFI) by flow cytometry. ^** p <0.01, ^* p <0.05 (calculated by unpaired t-test). Control antibody dosing in this experiment was 10 mg / kg (intravenous) initial dose on day 1 followed by 5 mg / kg (intraperitoneal) BIW. Anti-OX40 agonist antibody was dosed on day 1 at the initial dose of 0.1 mg / kg (intravenous) followed by 0.1 mg / kg (intraperitoneal) TIW. Treatment with anti-OX40 agonist antibody increased PDL1 expression in intratumoral myeloid (CD11b + Gr-1 low / intermediate) cells and (FIG. 3B) tumor cells in the CT26 colorectal syngeneic tumor model. Data are from 9 days after the start of treatment. Each dot / square represents one individual mouse. PDL1 expression measured by geometric mean fluorescence intensity (geometric MFI) by flow cytometry. ^** p <0.01, ^* p <0.05 (calculated by unpaired t-test). Control antibody dosing in this experiment was 10 mg / kg (intravenous) initial dose on day 1 followed by 5 mg / kg (intraperitoneal) BIW. Anti-OX40 agonist antibody was dosed on day 1 at the initial dose of 0.1 mg / kg (intravenous) followed by 0.1 mg / kg (intraperitoneal) TIW. Treatment with anti-OX40 agonist antibody and anti-PDL1 antagonist antibody demonstrated synergistic combined efficacy of MC38 colorectal cancer syngeneic tumor model in C57BL / 6 mice. (FIG. 4A) Measurement results of the average tumor volume (mm3) over time (daily) for each treatment group (n = 10 / group). The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Control antibody, anti-PDL1 antibody, or anti-OX40 agonist antibody was dosed on day 1 at the initial dose of 10 mg / kg (intravenous) followed by 10 mg / kg (intraperitoneal) TIW for 3 weeks. Treatment with anti-OX40 agonist antibody and anti-PDL1 antagonist antibody demonstrated synergistic combined efficacy of MC38 colorectal cancer syngeneic tumor model in C57BL / 6 mice. (FIG. 4B) Measurement results of individual tumor volumes over time for each treatment group. The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Control antibody, anti-PDL1 antibody, or anti-OX40 agonist antibody was dosed on day 1 at the initial dose of 10 mg / kg (intravenous) followed by 10 mg / kg (intraperitoneal) TIW for 3 weeks. Treatment with anti-OX40 agonist antibody and anti-PDL1 antagonist antibody demonstrated synergistic combined efficacy of CT26 colorectal syngeneic tumor model in Balb / c mice. (FIG. 5A) Measurement results of mean tumor volume (mm3) over time (daily) for each treatment group (n = 10 / group). (FIG. 5B) Measurement results of individual tumor volumes over time for each treatment group. Control antibody or anti-PDL1 was dosed on day 1 with an initial dose of 10 mg / kg (intravenous) followed by 5 mg / kg (intraperitoneal) TIW for 3 weeks. Anti-OX40 agonist antibody was administered at 1 mg / kg (intravenous) on day 1 as a single dose. The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Treatment with anti-OX40 agonist antibody and anti-PDL1 antagonist antibody demonstrated synergistic combined efficacy of CT26 colorectal syngeneic tumor model in Balb / c mice. (FIG. 5A) Measurement results of mean tumor volume (mm3) over time (daily) for each treatment group (n = 10 / group). (FIG. 5B) Measurement results of individual tumor volumes over time for each treatment group. Control antibody or anti-PDL1 was dosed on day 1 with an initial dose of 10 mg / kg (intravenous) followed by 5 mg / kg (intraperitoneal) TIW for 3 weeks. Anti-OX40 agonist antibody was administered at 1 mg / kg (intravenous) on day 1 as a single dose. The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Anti-OX40 agonist antibody single treatment shows the dose response of the CT26 colorectal cancer syngeneic tumor model in Balb / c mice. (FIG. 6A) Measurement results of mean tumor volume (mm3) over time (daily) for each treatment group (n = 10 / group). The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Control antibody was dosed on day 1 at the initial dose of 1 mg / kg (intravenous) followed by 1 mg / kg (intraperitoneal) TIW for 3 weeks. Anti-OX40 agonist antibody was dosed on day 1 at the initial dose of 0.01 mg / kg, 0.1 mg / kg, or 1 mg / kg (intravenous) followed by TIW (intraperitoneal) for 3 weeks. Anti-OX40 agonist antibody single treatment shows the dose response of the CT26 colorectal cancer syngeneic tumor model in Balb / c mice. (FIG. 6B) Measurement results of individual tumor volumes over time for each treatment group. The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Control antibody was dosed on day 1 at the initial dose of 1 mg / kg (intravenous) followed by 1 mg / kg (intraperitoneal) TIW for 3 weeks. Anti-OX40 agonist antibody was dosed on day 1 at the initial dose of 0.01 mg / kg, 0.1 mg / kg, or 1 mg / kg (intravenous) followed by TIW (intraperitoneal) for 3 weeks. The combined treatment of sub-maximal dose of anti-OX40 agonist antibody plus anti-PDL1 antagonist antibody demonstrated the synergistic combined efficacy of CT26 colorectal cancer syngeneic tumor model in Balb / c mice. (FIG. 7A) Measurement results of mean tumor volume (mm3) over time (daily) for each treatment group (n = 10 / group). The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Control antibody or anti-PDL1 was dosed on day 1 at the initial dose of 10 mg / kg (intravenous) followed by 10 mg / kg (intraperitoneal) TIW for 3 weeks. Anti-OX40 agonist antibody was administered on day 1 at the initial dose of 0.1 mg / kg (intravenous) followed by 0.1 mg / kg (intraperitoneal) TIW for 3 weeks. The combined treatment of sub-maximal dose of anti-OX40 agonist antibody plus anti-PDL1 antagonist antibody demonstrated the synergistic combined efficacy of CT26 colorectal cancer syngeneic tumor model in Balb / c mice. (FIG. 7B) Measurement results of individual tumor volumes over time for each treatment group. The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Control antibody or anti-PDL1 was dosed on day 1 at the initial dose of 10 mg / kg (intravenous) followed by 10 mg / kg (intraperitoneal) TIW for 3 weeks. Anti-OX40 agonist antibody was administered on day 1 at the initial dose of 0.1 mg / kg (intravenous) followed by 0.1 mg / kg (intraperitoneal) TIW for 3 weeks. In a separate experiment, CT26 colorectal syngeneic tumor model in Balb / c mice was administered by anti-OX40 agonist antibody plus anti-PDL1 in a single sub-maximal effective dose of 0.1 mg / kg (intravenous) infusion. Synergistic combination efficacy was demonstrated. (FIG. 8A) Measurement results of average tumor volume (mm3) over time (daily) for each treatment group (n = 10 / group). The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Control antibody or anti-PDL1 was dosed on day 1 with an initial dose of 10 mg / kg (intravenous) followed by 5 mg / kg (intraperitoneal) TIW for 3 weeks. Anti-OX40 antibody was administered as a first dose or a single dose of 0.1 mg / kg (intravenous) on day 1, followed by 0.1 mg / kg (intraperitoneal) TIW for 3 weeks. In a separate experiment, CT26 colorectal syngeneic tumor model in Balb / c mice was administered by anti-OX40 agonist antibody plus anti-PDL1 in a single sub-maximal effective dose of 0.1 mg / kg (intravenous) infusion. Synergistic combination efficacy was demonstrated. (FIG. 8B) Measurement results of individual tumor volumes over time for each treatment group. The black line shows the average of the treatment group. The blue dashed line shows the average of the control group. Gray lines are individual animals. The red line indicates individual animals that have been removed from the study due to tumor ulceration or excessive tumor size. Control antibody or anti-PDL1 was dosed on day 1 with an initial dose of 10 mg / kg (intravenous) followed by 5 mg / kg (intraperitoneal) TIW for 3 weeks. Anti-OX40 antibody was administered as a first dose or a single dose of 0.1 mg / kg (intravenous) on day 1, followed by 0.1 mg / kg (intraperitoneal) TIW for 3 weeks. Effect of combined treatment of OX40 agonist antibody and PDL1 antagonist (anti-PDL1 antagonist antibody) on proliferating T cells, Treg cells, plasma interferon-gamma, and activated T cell levels in peripheral blood. Analysis of peripheral blood collected from co-treated CT26 mice revealed an increase in effector cell proliferation and inflammatory T cell markers. The proliferation levels of CD8 + T cells (FIG. 9A), Treg cells (FIG. 9B), plasma interferon gamma levels (FIG. 9C), and activated T cells (FIG. 9D) were tested. (FIG. 9A) Proliferating CD8 + T cell levels (expressed as a percentage of ki67 + / total CD8 + T cells) are treated with OX40 agonist antibody and PD-L1 antagonist for animals treated with OX40 agonist antibody or PDL1 antagonist antibody alone. Marked increase in the animals. Effect of combined treatment of OX40 agonist antibody and PDL1 antagonist (anti-PDL1 antagonist antibody) on proliferating T cells, Treg cells, plasma interferon-gamma, and activated T cell levels in peripheral blood. Analysis of peripheral blood collected from co-treated CT26 mice revealed an increase in effector cell proliferation and inflammatory T cell markers. The proliferation levels of CD8 + T cells (FIG. 9A), Treg cells (FIG. 9B), plasma interferon gamma levels (FIG. 9C), and activated T cells (FIG. 9D) were tested. (FIG. 9B) Reduction of peripheral blood Treg was observed with treatment with OX40 agonist antibody alone and combined treatment with OX40 agonist antibody and PDL1 antagonist. Effect of combined treatment of OX40 agonist antibody and PDL1 antagonist (anti-PDL1 antagonist antibody) on proliferating T cells, Treg cells, plasma interferon-gamma, and activated T cell levels in peripheral blood. Analysis of peripheral blood collected from co-treated CT26 mice revealed an increase in effector cell proliferation and inflammatory T cell markers. The proliferation levels of CD8 + T cells (FIG. 9A), Treg cells (FIG. 9B), plasma interferon gamma levels (FIG. 9C), and activated T cells (FIG. 9D) were tested. (FIG. 9C) Increased plasma gamma interferon (IFNg) was observed with the combined treatment of OX40 agonist and PDL1 antagonist. Effect of combined treatment of OX40 agonist antibody and PDL1 antagonist (anti-PDL1 antagonist antibody) on proliferating T cells, Treg cells, plasma interferon-gamma, and activated T cell levels in peripheral blood. Analysis of peripheral blood collected from co-treated CT26 mice revealed an increase in effector cell proliferation and inflammatory T cell markers. The proliferation levels of CD8 + T cells (FIG. 9A), Treg cells (FIG. 9B), plasma interferon gamma levels (FIG. 9C), and activated T cells (FIG. 9D) were tested. (FIG. 9D) Animals whose levels of activated T cells (specifically, activated memory Teff cells) were treated with OX40 agonist antibody and PD-L1 antagonist in combination with treatment with OX40 agonist or PDL1 antagonist alone. Markedly increased. FIG. 5 shows the relationship between OX40 expression and PDL1 diagnostic status in cancer samples from human patients with urothelial bladder cancer (UBC, FIG. 10A) and non-small cell lung cancer (NSCLC, FIG. 10B). The tissue sample was from a patient who participated in a phase 1 clinical trial using the anti-PD-L1 antibody MPDL3280A. The IHC disclosed herein was used to determine the PD-L1 biomarker status of tumor infiltrating immune cells (IC). The OX40 expression level was determined using rtPCR analysis (Fluidigm). The triangle means that the patient showed a partial or complete clinical response, the circle means that the patient showed stable disease, and the square means that the patient showed progressive disease To do. FIG. 5 shows the relationship between OX40 expression and PDL1 diagnostic status in cancer samples from human patients with urothelial bladder cancer (UBC, FIG. 10A) and non-small cell lung cancer (NSCLC, FIG. 10B). The tissue sample was from a patient who participated in a phase 1 clinical trial using the anti-PD-L1 antibody MPDL3280A. The IHC disclosed herein was used to determine the PD-L1 biomarker status of tumor infiltrating immune cells (IC). The OX40 expression level was determined using rtPCR analysis (Fluidigm). The triangle means that the patient showed a partial or complete clinical response, the circle means that the patient showed stable disease, and the square means that the patient showed progressive disease To do. 2 shows an exemplary IHC analysis of a control cell sample. (FIG. 11A) Negative control IHC staining of parent HEK-293 cells, (FIG. 11B) IHC staining at low staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (FIG. 11C) Recombinant human PD -IHC staining with moderate staining intensity of HEK-293 cells transfected with L1, (Fig. 11D) IHC staining with strong staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (Fig. 11E) Positive tissue control IHC staining of placental tissue samples, (FIG. 11F) Positive tissue control IHC staining of tonsil tissue samples. All IHC staining was performed using a proprietary anti-PD-L1 antibody. 2 shows an exemplary IHC analysis of a control cell sample. (FIG. 11A) Negative control IHC staining of parent HEK-293 cells, (FIG. 11B) IHC staining at low staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (FIG. 11C) Recombinant human PD -IHC staining with moderate staining intensity of HEK-293 cells transfected with L1, (Fig. 11D) IHC staining with strong staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (Fig. 11E) Positive tissue control IHC staining of placental tissue samples, (FIG. 11F) Positive tissue control IHC staining of tonsil tissue samples. All IHC staining was performed using a proprietary anti-PD-L1 antibody. 2 shows an exemplary IHC analysis of a control cell sample. (FIG. 11A) Negative control IHC staining of parent HEK-293 cells, (FIG. 11B) IHC staining at low staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (FIG. 11C) Recombinant human PD -IHC staining with moderate staining intensity of HEK-293 cells transfected with L1, (Fig. 11D) IHC staining with strong staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (Fig. 11E) Positive tissue control IHC staining of placental tissue samples, (FIG. 11F) Positive tissue control IHC staining of tonsil tissue samples. All IHC staining was performed using a proprietary anti-PD-L1 antibody. 2 shows an exemplary IHC analysis of a control cell sample. (FIG. 11A) Negative control IHC staining of parent HEK-293 cells, (FIG. 11B) IHC staining at low staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (FIG. 11C) Recombinant human PD -IHC staining with moderate staining intensity of HEK-293 cells transfected with L1, (Fig. 11D) IHC staining with strong staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (Fig. 11E) Positive tissue control IHC staining of placental tissue samples, (FIG. 11F) Positive tissue control IHC staining of tonsil tissue samples. All IHC staining was performed using a proprietary anti-PD-L1 antibody. 2 shows an exemplary IHC analysis of a control cell sample. (FIG. 11A) Negative control IHC staining of parent HEK-293 cells, (FIG. 11B) IHC staining at low staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (FIG. 11C) Recombinant human PD -IHC staining with moderate staining intensity of HEK-293 cells transfected with L1, (Fig. 11D) IHC staining with strong staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (Fig. 11E) Positive tissue control IHC staining of placental tissue samples, (FIG. 11F) Positive tissue control IHC staining of tonsil tissue samples. All IHC staining was performed using a proprietary anti-PD-L1 antibody. 2 shows an exemplary IHC analysis of a control cell sample. (FIG. 11A) Negative control IHC staining of parent HEK-293 cells, (FIG. 11B) IHC staining at low staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (FIG. 11C) Recombinant human PD -IHC staining with moderate staining intensity of HEK-293 cells transfected with L1, (Fig. 11D) IHC staining with strong staining intensity of HEK-293 cells transfected with recombinant human PD-L1, (Fig. 11E) Positive tissue control IHC staining of placental tissue samples, (FIG. 11F) Positive tissue control IHC staining of tonsil tissue samples. All IHC staining was performed using a proprietary anti-PD-L1 antibody. FIG. 12A shows exemplary PD-L1-positive IHC staining of a tumor sample from triple negative breast cancer, (FIG. 12B) malignant melanoma, (FIG. 12C) NSCLC, adenocarcinoma. FIG. 12A shows exemplary PD-L1-positive IHC staining of a tumor sample from triple negative breast cancer, (FIG. 12B) malignant melanoma, (FIG. 12C) NSCLC, adenocarcinoma. FIG. 12A shows exemplary PD-L1-positive IHC staining of a tumor sample from triple negative breast cancer, (FIG. 12B) malignant melanoma, (FIG. 12C) NSCLC, adenocarcinoma.

  The inventors of the present application have demonstrated that combined immunotherapy of anti-human OX40 agonist antibody and anti-PD-L1 results in synergistic inhibition of tumor growth and increased response rate.

  In one aspect, methods, compositions, and uses for treating cancer or delaying its progression in an individual comprising administering an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist. Provided in the description.

  In another aspect, methods, compositions, and uses herein for enhancing immune function in an individual with cancer comprising administering an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist. Provided.

  In another aspect, to treat an infection (eg, a bacterial or viral infection or other pathogen infection) in an individual with cancer comprising administering an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist. Methods, compositions, and uses are provided herein.

I. Definitions Before describing the present invention in detail, it is to be understood that the present invention is not limited to a particular composition or biological system, but of course it is diverse. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

  As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and so forth.

  As used herein, the term “about” refers to the normal error range for each value as would be readily understood by one skilled in the art. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter itself.

  It is understood that aspects and embodiments of the invention described herein include “comprising”, “consisting of”, and “consisting essentially of” aspects and embodiments.

The term “OX40” as used herein is from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise indicated. Refers to any natural OX40. The term encompasses “full-length” unprocessed OX40, as well as any form of OX40 resulting from processing in a cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants. The amino acid sequence of an exemplary human OX40 lacking a signal peptide is shown in SEQ ID NO: 60.

  “OX40 activation” refers to activation of the OX40 receptor. In general, OX40 activation results in signal transduction.

The terms “anti-OX40 antibody” and “antibody that binds to OX40” refer to binding to OX40 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting OX40. It refers to an antibody that can. In one embodiment, the degree of binding of the anti-OX40 antibody to an unrelated non-OX40 protein is less than about 10% of the binding of the antibody to OX40, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to OX40 is 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (eg, 10 ⁻⁸ M or less, For example, it has a dissociation constant (Kd) of 10 ⁻⁸ M to 10 ⁻¹³ M, eg, 10 ⁻⁹ M to 10 ⁻¹³ M). In certain embodiments, the anti-OX40 antibody binds to an epitope of OX40 that is conserved among OX40 from different species.

  The term “PD-1 axis binding antagonist” refers to any one of the PD-1 axis binding partner and its binding partner so as to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis. Refers to a molecule that inhibits interaction with one or more and consequently restores or enhances T cell function (eg, proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

  The term “PD-1 binding antagonist” refers to reducing, blocking, inhibiting signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, eg, PD-L1, PD-L2. Refers to a molecule that inhibits or interferes. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In one specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists are due to anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and the interaction of PD-1 with PD-L1 and / or PD-L2. Other molecules that reduce, block, inhibit, suppress, or interfere with signal transduction. In one embodiment, the PD-1 binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed in T lymphocytes that mediated signaling through PD-1. Make dysfunctional T cells dysfunctional lower (eg, enhance effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In one specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab) as described herein. In another specific aspect, the PD-1 binding antagonist is MK-3475 (Pembrolizumab) as described herein. In another specific embodiment, the PD-1 binding antagonist is CT-011 (pizilizumab) as described herein. In another specific aspect, the PD-1 binding antagonist is AMP-224 as described herein.

  The term “PD-L1 binding antagonist” reduces, blocks signal transduction due to interaction of PD-L1 with any one or more of its binding partners, eg PD-1, B7-1, Refers to a molecule that inhibits, suppresses, or interferes with. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In one specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, and one or more of PD-L1 and its binding partner, For example, other molecules that reduce, block, inhibit, suppress, or interfere with signal transduction due to interaction with PD-1, B7-1. In one embodiment, the PD-L1 binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed in T lymphocytes that mediated signaling via PD-L1, Make dysfunctional T cells dysfunctional lower (eg, enhance effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In one specific aspect, the anti-PD-L1 antibody is YW243.55. S70. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 as described herein. In yet another specific aspect, the anti-PD-L1 antibody is MPDL3280A as described herein. In yet another specific aspect, the anti-PD-L1 antibody is MEDI4736 as described herein.

  The term “PD-L2 binding antagonist” refers to reducing, blocking, inhibiting, deterring signal transduction due to interaction with any one or more of PD-L2 and its binding partners, eg PD-1. Or refers to interfering molecules. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In one specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonist is any one or more of an anti-PD-L2 antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, and PD-L2 and its binding partner. For example, including other molecules that reduce, block, inhibit, suppress, or interfere with signal transduction resulting from interaction with PD-1. In one embodiment, the PD-L2 binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed in T lymphocytes that mediated signal transduction via PD-L2, Make dysfunctional T cells dysfunctional lower (eg, enhance effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

  The term “dysfunction” in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes common elements of both wasting and / or anergy where antigen recognition can occur, but subsequent immune responses are ineffective in controlling infection or tumor growth.

  As used herein, the term “dysfunction” refers to refractory or unresponsiveness to antigen recognition, specifically, antigen recognition down-stream T cell effector functions such as proliferation, cytokine production (eg IL -2), and / or impaired ability to translate into target cell death.

The term “anergy” refers to a state of unresponsiveness to antigenic stimulation (eg, intracellular Ca in the absence of Ras activation due to an incomplete or insufficient signal delivered via the T cell receptor. ⁺² increase). T cell anergy can also occur upon stimulation with an antigen in the absence of costimulation, resulting in refractory to subsequent activation by the antigen, even in the context of costimulation. The unresponsive state can often be overridden by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and / or do not acquire effector function.

  The term “depletion” refers to T cell depletion as a state of T cell dysfunction resulting from many chronic infections and persistent TCR signaling that occurs during cancer development. This is distinguished from anergy in that it is due to sustained signaling rather than incomplete or insufficient signaling. This is defined by effector dysfunction distinct from functional effector or memory T cells, persistent expression of inhibitory receptors, and transcriptional status. Exhaustion prevents optimal control of infection and tumor. Depletion is in both extrinsic negative regulatory pathways (eg, immunoregulatory cytokines) and intracellular intrinsic negative regulatory (costimulatory) pathways (PD-1, B7-H3, B7-H4, etc.) Can be attributed.

  “Enhance T cell function” refers to inducing, causing or stimulating T cells to have sustained or amplified biological function, or exhausted or inactive T cells Means to renew or reactivate. Examples of enhanced T cell function include increased secretion of gamma-interferon from CD8 + T cells, increased proliferation, increased antigen responsiveness compared to pre-intervention levels (eg, virus, pathogen, or tumor clearance). Can be mentioned. In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner in which this enhancement is measured is known to those skilled in the art.

  A “T cell dysfunction disorder” is a T cell disorder or condition characterized by a decreased responsiveness to antigenic stimulation. In one specific embodiment, the T cell dysfunction disorder is a disorder particularly associated with increased inappropriate signaling through PD-1. In another embodiment, a T cell dysfunction disorder is a disorder in which T cells are anergic or have a reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In one specific aspect, the reduced responsiveness results in ineffective control of the pathogen or tumor that expresses the immunogen. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

  “Tumor immunity” refers to the process by which a tumor avoids immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is “treated” when such evasion is attenuated and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor contraction, and tumor clearance.

  “Immunogenic” refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and the enhanced tumor immunogenicity assists in the clearance of tumor cells by the immune response. Examples of enhanced tumor immunogenicity include treatment with a PD-1 axis binding antagonist and an OX40 binding agonist.

  “Sustained response” refers to the sustained effect on reducing tumor growth after cessation of treatment. For example, the tumor size can remain the same or smaller compared to the size at the beginning of the dosing phase. In some embodiments, the sustained response has a period at least as long as the treatment period, at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times the treatment period.

  The term “pharmaceutical formulation” refers to a preparation that is in a form such that the biological activity of the active ingredient is effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those that can be moderately administered to a subject mammal to provide an effective dose of the active ingredient to be used.

  As used herein, the term “treatment” refers to a clinical intervention designed to change the natural course of an individual or cell being treated during the course of a clinical lesion. Desirable effects of treatment include reduction of disease progression rate, recovery or alleviation of disease state, and remission or improvement of prognosis. For example, an individual may be able to reduce (or destroy) the growth of cancerous cells, reduce symptoms due to the disease, improve the quality of life of those suffering from the disease, and doses of other medications needed to treat the disease. Is successfully “treated” if one or more symptoms associated with cancer are reduced or eliminated, including, but not limited to, reducing and / or prolonging the survival of an individual.

  As used herein, “slowing the progression of a disease” means delaying, impeding, slowing, delaying, stabilizing, and / or prolonging the onset of a disease (such as cancer). . This delay can be of varying duration depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can effectively encompass prophylaxis in that the individual does not develop the disease. For example, late-stage cancer such as onset of metastasis can be delayed

  An “effective amount” is at least the minimum amount necessary to achieve a measurable improvement or prevention of a particular disorder. Effective amounts herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit the desired response in the individual. An effective amount is also one in which the therapeutically beneficial effect exceeds any toxic or adverse effects of the treatment. For prophylactic use, beneficial or desired outcomes include biochemical, histological and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the onset of the disease. Results include elimination or reduction of disease risk, reduction of disease severity, or delay of disease development. For therapeutic use, beneficial or desired results include reduction of one or more symptoms resulting from the disease, improvement of the quality of life of those suffering from the disease, other medications necessary for the treatment of the disease Clinical outcomes such as reducing the dose of another drug, enhancing the effect of another drug (eg, by target), delaying disease progression, and / or prolonging survival. In the case of a cancer or tumor, an effective amount of drug reduces the number of cancer cells, reduces tumor size, and inhibits cancer cells from invading peripheral organs (ie, delayed to some extent or desirably stopped). May have the effect of inhibiting tumor metastasis (ie, delaying or desirably stopping to some extent), inhibiting tumor growth to some extent, and / or reducing one or more of the symptoms associated with the disorder to some extent . An effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment either directly or indirectly. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” can be considered in the context of administration of one or more therapeutic agents, and a single agent can be combined with one or more other agents to achieve or achieve the desired result. Can be considered to be given in an effective amount.

  As used herein, “in conjunction with” refers to the administration of another therapy in addition to one therapy. Thus, "in conjunction with" refers to the administration of another treatment before, during, or after administration of one treatment to an individual.

  A “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. .

  The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

  “Tumor” as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder”, and “tumor” are not mutually exclusive as referred to herein.

  The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy. More specific examples of such cancer include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer, eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocyte Cancer, gastric cancer or stomach cancer, eg, gastrointestinal and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer Breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma , Superficial enlarged melanoma, malignant melanoma, terminal melanoma, nodular melanoma, multiple myeloma and B cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphoma Spherical (SL) NHL, intermediate grade / follicle NHL, moderate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleavable NHL, large lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, And Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder ( PTLD), as well as abnormal blood vessel growth associated with nevus, edema (such as that associated with brain tumors), Megs syndrome, brain cancer, and head and neck cancer, and related metastases, but are not limited to these. In certain embodiments, cancers amenable to treatment with an antibody of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin lymphoma (NHL), renal cell carcinoma Prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi sarcoma, carcinoid cancer, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Further, in some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (including these metastatic forms) cancer.

The term “cytotoxic agent” as used herein refers to any agent that is detrimental to cells (eg, causes cell death, inhibits proliferation, or otherwise interferes with cell function). . Cytotoxic agents include radioisotopes (eg, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and radioisotope Lu), chemical Therapeutic agents, growth inhibitors, enzymes and fragments thereof, such as nucleolytic enzymes, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (fragments and / or variants thereof) Including, but not limited to, shapes). Exemplary cytotoxic agents include anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors , Non-receptor tyrosine kinase angiogenesis inhibitor, immunotherapeutic agent, apoptosis promoter, LDH-A inhibitor, fatty acid biosynthesis inhibitor, cell cycle signaling inhibitor, HDAC inhibitor, proteasome inhibitor, and cancer metabolism inhibition Agents can be selected. In one embodiment, the cytotoxic agent is a taxane. In one embodiment, the taxane is paclitaxel or docetaxel. In one embodiment, the cytotoxic agent is a platinum agent. In one embodiment, the cytotoxic agent is an antagonist of EGFR. In one embodiment, the antagonist of EGFR is N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) quinazolin-4-amine (eg, erlotinib). In one embodiment, the cytotoxic agent is a RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and / or CRAF inhibitor. In one embodiment, the RAF inhibitor is vemurafenib. In one embodiment, the cytotoxic agent is a PI3K inhibitor.

  A “chemotherapeutic agent” includes compounds that are useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib 17 -AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX (registered trademark), AstraZeneca), Sunitib (SUTENT (registered trademark), Pfizer / Sugen), letrozole (FEMARA ( (Registered trademark), Novartis), imatinib mesylate (GLEEVEC (registered trademark), Novartis), finasnate (VATALANIB (registered trademark)) , Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®), GSK572016, Glaxo Smith Kline), Lonafarmib (SCH 66336), Sorafenib (NEXAVAR (R), Bayer Labs), Gefitinib (IRESSA (R), AstraZeneca), AG1478, alkylating agents such as Tiotepa and CYTOX Phosphamide, alkyl sulfonates such as busulfan, improsulfan, and pipersulfan, aziridine, eg For example, benzodopa, carbocon, metredorpa, and ureodopa, ethyleneimine and methylalamine, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine, acetogenin (especially bratacin and bratacinone), camptothecin (Such as topotecan and irinotecan), bryostatin, calistatin, CC-1065 (such as its adzelesin, calzeresin, and biselecin synthetic analogs), cryptophysin (especially cryptophysin 1 and cryptophysin 8), corticosteroids (prednisone and prednisolone) ), Cyproterone acetate, 5α-reductase such as finasteride and dutasteride), vorinostat, romidepsin, Binostat, valproic acid, mocetinostat dolastatin, aldesleukin, duocarmycin (such as the synthetic analogues KW-2189 and CB1-TM1), eluterobin, pancratistatin, sarcosine, spongistatin, nitrogen mustard, for example , Chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitine, phenesterine, prednimustine, trophosphamide, uracil mustard, nitrosourea, such as carmustine, chlorozotocin, Fotemustine, lomustine, nimustine, and ranimustine, antibiotics such as enediyne antibiotics (eg calicheamicin, Calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994 33: 183-186), dynemicins, such as dynemicin A, bisphosphonates, such as clodronate, esperamicin, and the neocartinostatin and related chromoprotein energin antibiotic luminophores), aclacinomycin, actinomycin, ausula Mycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin) Morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxyxorubicin), epirubicin, eso Bicine, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olivomycin, pepromycin, porphyromycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin Antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogues such as denopterine, methotrexate, pteropterin, trimetrexate, purine analogues such as fludarabine, 6-mercaptopurine, thiapurine, thioguanine , Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxy Uridine, doxyfluridine, enocitabine, furoxyuridine, androgen, such as carsterone, drmostanolone propionate, epithiostanol, mepithiostan, test lactone, anti-adrenal, such as aminoglutethimide, mitotane, trilostane, folic acid supplement, such as floric acid , Acegraton, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, vestlabsyl, bisantrene, edatralxate, defofamine, demecorsin, diazicon, erfornitine, ellipticine acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan Lonidamine, maytansinoids such as maytansine and ansamitocin, mitguazone, mitoxantrone, D'Amour, Nitorakurin, pentostatin, Fenametto, pirarubicin, losoxantrone, podophyllin acid, 2-ethyl-hydrazide, procarbazine, PSK (R) polysaccharide complex (JHS Natural Products, Eugene, Oreg. ), Razoxan, lysoxine, schizophyllan; spirogermanium, tenuazonic acid, triadicon, 2,2 ′, 2 ″ -trichlorotriethylamine, trichothecene (especially T-2 toxin, veraculin A, loridene A, and anguidene), urethane, vindesine , Dacarbazine, mannomustine, mitoblonitol, mitactol, piperobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxoid, such as TAXOL (paclitaxel, Bristol-Myers Squibb, Nc. , ABRAXANE® (without cremophor), paclitaxel albumin engineered nanoparticle formulation (American Pharmace tical Partners, Schaumberg, Ill.), and TAXOTERE® (docetaxel, doxetaxel, Sanofi-Aventis), chlorambucil, GEMZAR® (gencitabine), 6-thioguanine, mercaptopurine, platinum analog Cisplatin and carboplatin, vinblastine, etoposide (VP-16), ifosfamide, mitoxantone, vincristine, NAVELBINE® (vinorelbine), novantrone, teniposide, edatrexate, daunomycin, aminopterin, capecitabine (XELODA) Ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylo Examples include ernitine (DMFO), retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

  Chemotherapeutic agents include (i) antihormonal agents that serve to control or inhibit hormonal effects on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (NOLVADEX®). Tamoxifen citrate, etc.), raloxifene, droloxifene, iodoxifene, 4-hydroxy tamoxifen, trioxyphene, keoxifene, LY11018, onapristone, FARESTON (registered trademark) (toremifene citrate), etc. (ii) ) Aromatase inhibitors that inhibit the enzyme aromatase that controls estrogen production in the adrenal glands, such as 4 (5) -imidazole, aminoglutethimide, MEGASE® (megesterol acetate), AROM SIN (R) (Exemestane, Pfizer), Formestane, Fadrozole, RIVISOR (R) (borozole), FEMARA (R) (Letrozole, Novartis), ARIMIDEX (R) Anastrozole, AstraZeneca, etc. (Iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all trans retinoic acid, fenretinide, and troxacitabine (1,3-dioxirane nucleoside cytosine analog), (iv) protein kinase inhibitor, (v) lipid kinase inhibitor, vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell growth, such as PKC-alpha, Ralf, and H-Ras, etc. (vii) ribozymes, such as VEGF Expression inhibitors (eg ANGIOZYME®) and HER2 expression inhibitors, etc. (viii) vaccines, eg gene therapy vaccines, eg ALLOVECTIN®, LEUVECTIN®, and VAXID® , PROLEUKIN®, rIL-2, topoisomerase 1 inhibitor, such as LURTOTECAN®, ABARELIX® rmRH, and (ix) a pharmaceutically acceptable salt of any of the above, Acids and derivatives Also mentioned.

Chemotherapeutic agents include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech), cetuximab (ERBITUX®, Imclone), panitumumab (VECTIBIX®, Amgen), rituximab RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia conjugate), and antibody-drug Also included is gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Further humanized monoclonal antibodies having therapeutic potential as a drug in combination with the compounds of the present invention include apolizumab, acelizumab, atlizumab, bapineuzumab, bibatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pe Gol, Sidofizumab, Sidotuzumab, Daclizumab, Ecilizumab, Ecilizumab, Elizizumab, Elizizumab, Elizizumab, Epiratuzumab, Ellizumab, Felbizumab, Fontlizumab, Gemtuzumab Ozogamicin, Inotuzumab Motobizumab, natalizumab, nimotuzumab, norobizumab, numabizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pekfushizumab, Kutzuzumab, Pexelizumab, Laribizumab, Ranibizumab, Lesribizumab, Leslizumab, Resibizumab, Rovelizumab, Ruprizumab, Cibrotuzumab, Ciprizumab, Sontuzumab, Tacuzumab Tetrazetane, Tadozumab Full length of recombinant human sequences, genetically modified to recognize interleukin-12 p40 protein, Ulutoxazumab, ustekinumab, bicilizumab, and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories) IgG ₁ λ antibody).

  A chemotherapeutic agent refers to a compound that binds to or otherwise interacts directly with EGFR and prevents or reduces its signaling activity, including "EGFR inhibitors", also referred to as "EGFR antagonists". It is done. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (US Pat. No. 4,943). 533 (Mendelsohn et al.), And variants thereof, such as chimerization 225 (C225 or cetuximab, ERBUIX®) and reshaped human 225 (H225) (WO 96/40210 (Imclone)). Systems Inc.), IMC-11F8, fully human EGFR targeting antibody (Imclone), antibodies that bind to type II mutant EGFR (US Pat. No. 5,212,290), US Pat. No. 5,891 , 996, humanized and chimeric antibodies that bind to EGFR, and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98/50433, Abgenix / Amgen), EMD55900 (Stragliototo et al. al.Eur.J. Cancer 32A: 636-640 (1996)), a humanized EGFR antibody EMD7200 (matsuzumab) directed against EGFR that competes with both EGF and TGF-alpha for EGFR binding (EMD / Merck). Known as the human EGFR antibody HuMax-EGFR (GenMab), E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3, And in US 6,235,883 Mounting the fully human antibody, MDX-447 (Medarex Inc), as well as mAb806 or humanized mAb806 (Johns et al, J.Biol.Chem.279 (29):. 30375-30384 (2004)) and the like. Anti-EGFR antibodies can be conjugated to cytotoxic drugs and thus generate immunoconjugates (see, eg, EP659439A2 (Merck Patent GmbH)). EGFR antagonists are described in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713 484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747 , 498, and the following PCT publications WO 98/14451, WO 98/5 038, to WO99 / 09016, and WO99 / 24 037 comprises a small molecule, such as the compounds described. Specific small molecule EGFR antagonists include OSI-774 (CP-358774, Erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals), PD183805 (CI1033, 2-propenamide, N- [4-[(3-chloro -4-fluorophenyl) amino] -7- [3- (4-morpholinyl) propoxy] -6-quinazolinyl]-, dihydrochloride, Pfizer Inc.), ZD1839, gefitinib (IRESSA®) 4- ( 3′-chloro-4′-fluoroanilino) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, AstraZeneca), ZM105180 ((6-amino-4- (3-methylphenyl-amino) -quinazoline, Zene ca), BIBX-1382 (N8- (3-chloro-4-fluoro-phenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimido [5,4-d] pyrimidine-2,8-diamine) Boehringer Ingelheim), PKI-166 ((R) -4- [4-[(1-phenylethyl) amino] -1H-pyrrolo [2,3-d] pyrimidin-6-yl] -phenol), (R ) -6- (4-hydroxyphenyl) -4-[(1-phenylethyl) amino] -7H-pyrrolo [2,3-d] pyrimidine), CL-387785 (N- [4-[(3-bromo Phenyl) amino] -6-quinazolinyl] -2-butynamide), EKB-569 (N- [4-[(3-chloro-4-fluorophenyl) amino] -3-cyano-7-ethoxy 6-quinazolinyl] -4- (dimethylamino) -2-butynamide) (Wyeth), AG1478 (Pfizer), AG1571 (SU 5271, Pfizer), dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®) Trademark), GSK572016 or N- [3-chloro-4-[(3-fluorophenyl) methoxy] phenyl] -6 [5 [[[2-methylsulfonyl) ethyl] amino] methyl] -2-furanyl] -4 -Quinazolineamine).

  Chemotherapeutic agents include “tyrosine kinase inhibitors”, eg, the EGFR target drug described in the previous paragraph, small molecule HER2 tyrosine kinase inhibitors, eg, TAK165, CP-724, 714, ErbB2 receptor available from Takeda Oral selective inhibitors of tyrosine kinases (Pfizer and OSI), dual HER inhibitors such as EKB-569 (available from Wyeth, which binds preferentially to EGFR but inhibits both HER2 and EGFR overexpressing cells ), Lapatinib (available from GSK572016, Glaxo-SmithKline), oral HER2 and EGFR tyrosine kinase inhibitors, PKI-166 (available from Novartis), pan-HER inhibitors such as caneltinib (CI-1033, Pharm) cia), Raf-1 inhibitors such as ISIS Pharmaceuticals that inhibit Raf-1 signaling ISIS-5132, non-HER targeted TK inhibitors such as imatinib mesylate (GLEVEEC®) , Available from Glaxo SmithKline), multi-target tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer), VEGF receptor tyrosine kinase inhibitors such as bataranib (PTK787 / ZK222584, Novartis / Schering) Available from AG), MAPK extracellular regulatory kinase I inhibitor CI-1040 (available from Pharmacia), quinazolines such as PD 153035, 4- (3 -Chloroanilino) quinazoline, pyridopyrimidine, pyrimidopyrimidine, pyrrolopyrimidine, for example CGP 59326, CGP 60261, and CGP 62706, pyrazolopyrimidine, 4- (phenylamino) -7H-pyrrolo [2,3-d] pyrimidine , Curcumin (diferloylmethane, 4,5-bis (4-fluoroanilino) phthalimide), tyrphosti containing a nitrothiophene moiety, PD-0183805 (Warner-Lamber), antisense molecules (eg HER-encoding nucleic acids Quinoxaline (US Pat. No. 5,804,396), tyrphostin (US Pat. No. 5,804,396), ZD6474 (Astra Zeneca), PTK-787 (Novartis / Schering) AG), pan-HER inhibitors such as CI-1033 (Pfizer), Affinitac (ISIS 3521, Isis / Lilly), imatinib mesylate (GLEEVEC®), PKI166 (Novatis), GW2016 (GlaxoSmith), CI-1033 (Pfizer), EKB-569 (Wyeth), Semaxinib (Pfizer), ZD6474 (AstraZeneca), PTK-787 (Novartis / Schering AG), INC-1C11 (Imclone), Rapamycin (Sirolims E, AP) ), Or the following patent publications: US Pat. No. 5,804,396, WO 1999/09016 (American Cyanamid) , WO 1998/43960 (American Cyanamid), WO 1997/38983 (Warner Lambert), WO 1999/06378 (Warner Lambert), WO 1999/06396 (Warner Lambert), WO 1996/30967 (Inc3 / 1996) / 3397 (Zeneca) and those described in WO1996 / 33980 (Zeneca).

  As chemotherapeutic agents, dexamethasone, interferon, colchicine, metopurine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG raw, bevacizumab, bexarotene, cladribine, crofarine alfa Dexrazoxane, epoetin alfa, erlotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxalene, nandrolone, nelarabine, nofetumomab, oplerbequine demerzemer paliperomepamide Peg asparager , Pegfilgrastim, pemetrexed disodium, pricamycin, porfimer sodium, quinacrine, rasburicase, salgramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronic acid, and zoledronic acid Also included are pharmaceutically acceptable salts.

Chemotherapeutic agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, thixcortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amsinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone sodium phosphate, Dexamethasone, Dexamethasone sodium phosphate, fluocortron, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, alcromethasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17 -Propionate, fluocortron caproate, fluocortron pivalate, and acetate Prednidene, immunoselective anti-inflammatory peptide (ImSAID) such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN BioTherapeutics, LLC), anti-rheumatic drugs such as azathioprine, cyclosporine ( Cyclosporine A), D-penicillamine, gold salt, hydroxychloroquine, leflunomidominocycline, sulfasalazine, tumor necrosis factor affa (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab Gol (Cimzia), Golimumab (Simponi), Interleukin 1 (IL-1) blockers such as Anakinra, T Costimulatory blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®), interleukin 13 (IL-13) blockers such as lebrikizumab, interferon Alpha (IFN) blockers such as rontalizumab, beta7 integrin blockers such as rhuMAb beta7, IgE pathway blockers such as anti-M1 prime secreted homotrimeric LTa3 and membrane bound heterotrimeric LTal / β2 blockers such as anti-lymphotoxin alpha (LTa), radioisotopes (eg At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , And radioisotopes Lu), various treatments Drugs, for example, Chiopurachin, PS-341, phenylbutyrate, ET-18-OCH ₃ or farnesyl transferase inhibitors, (L-739749, L- 744832), a polyphenol, e.g., quercetin, resveratrol, piceatannol, Epigallocatechin gallate, theaflavin, flavanols, procyanidins, betulinic acid, and derivatives thereof, autophagy inhibitors such as chloroquine, delta-9-tetrahydrocannabinol (dronabinol, MARINOL®), beta-lapachone, Rapacol, colchicine, betulinic acid, acetylcamptothecin, scopoletin, and 9-aminocamptothecin), podophyllotoxin, tegafur (UFTORAL®), bexarotene TARGRETIN®), bisphosphonates such as clodronate (eg BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®) )), Alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®), and epidermal growth factor receptor (EGF) -R), vaccines such as THERATOPE® vaccine, perifosine, COX-2 inhibitors (eg celecoxib or etoroxib), proteosome inhibitors (eg PS3 1), CCI-779, tipifarnib (R11577), orafenib, ABT510, Bcl-2 inhibitors such as obrimersen sodium (GENAENSE®), pixantrone, farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASAR ( )), And pharmaceutically acceptable salts, acids, or derivatives of any of the above, and combinations of two or more of the above, eg, CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) Abbreviated combination therapy), and FOLFOX (abbreviation for treatment regimen in combination with OXARIplatin (ELOXATIN ™) and 5-FU and leucovorin).

  Chemotherapeutic agents also include non-steroidal anti-inflammatory drugs that have analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid. Derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoroxib, lumiracoxib, Examples include parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs are rheumatoid arthritis, osteoarthritis, inflammatory arthritis, ankylosing spondylitis, psoriatic arthritis, Reiter syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, postoperative pain, inflammation And may be needed for the relief of conditions such as mild to moderate pain due to tissue damage, fever, bowel obstruction, and renal colic.

  As used herein, a “growth inhibitory agent” refers to a compound or composition that inhibits cell growth either in vitro or in vivo. In one embodiment, the growth inhibitory agent is a growth inhibitory antibody that prevents or reduces the proliferation of cells that express the antigen to which the antibody binds. In another embodiment, the growth inhibitor may be one that significantly reduces the proportion of S phase cells. Examples of growth inhibitors include agents that block cell cycle progression (at places other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classic M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that stop G1, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also affect S phase arrest. For further information, see Murakami et al., Mendelsohn and Israel, eds. , The Molecular Basis of Cancer, Chapter 1, title “Cell cycle regulation, oncogenes, and antineoplastic drugs” (WB Saunders, Philadelphia, 1995), eg, 13 tributes. Taxanes (paclitaxel and docetaxel) are both yew-derived anticancer drugs. European Yew-derived docetaxel (TAXOTERE®, Rhone-Poulenc Roller) is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of tubulin dimer-derived microtubules, stabilize the microtubules by preventing depolymerization, and inhibit mitosis within the cell.

  “Radiation therapy” means the use of directed gamma or beta radiation to induce sufficient damage to cells to function normally or limit the ability to completely destroy the cells. It is understood that there are many methods known in the art for determining many dosages and duration of treatment. Typical treatment is given as a single dose, with typical dosages ranging from 10 to 200 units (gray) per day.

  “Subjects” or “individuals” for therapeutic purposes are classified into mammals such as humans, domestic animals and livestock, and zoo, sport or pet animals such as dogs, horses, cats, cows, etc. Refers to any animal. Preferably the mammal is a human.

  The term “antibody” in the present specification is used in the broadest sense, and specifically, as long as they exhibit a desired biological activity, monoclonal antibodies (such as full-length monoclonal antibodies), polyclonal antibodies, multispecificity Includes antibodies (eg, bispecific antibodies), and antibody fragments.

  An “isolated” antibody is an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that will interfere with the research, diagnostic, or therapeutic use of antibodies, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Can be mentioned. In some embodiments, the antibody is (1) greater than 95% by weight, for example, greater than 99% by weight, as determined by, for example, the Raleigh method, and in some embodiments greater than 99% by weight. Using a cup sequenator to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) reduced or non-reduced using, for example, Coomassie blue or silver stain Purify until homogeneity is obtained by SDS-PAGE under conditions. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

A “natural antibody” is a heterotetrameric glycoprotein of approximately 150,000 daltons, usually composed of two identical light (L) chains and two identical heavy (H) chains. While each light chain is linked to the heavy chain by one disulfide covalent bond, the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain (V _L ) on one end and a constant domain on the other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable The domain aligns with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

The term “constant domain” refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to other portions of an immunoglobulin that are variable domains containing an antigen binding site. The constant domains are heavy chain _C H _{1, C} H 2, and _{C H} 3 domains (collectively, CH), and contains a light chain CHL (or CL) domain.

The “variable region” or “variable domain” of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The heavy chain variable domain may be referred to as “V _H ”. The light chain variable domain may be referred to as “V _L ”. These domains are generally the most variable parts of the antibody and contain the antigen binding site.

  The term “variable” refers to the fact that the sequence of a particular portion of a variable domain varies widely between antibodies and is used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. This is concentrated in three segments called hypervariable regions (HVRs) in both the light chain variable domain and the heavy chain variable domain. The more highly conserved portions of variable domains are called the framework region (FR). Natural heavy and light chain variable domains each connect beta-sheet structures, and in some cases beta connected by three HVRs forming a loop that forms part of the beta-sheet structure Includes four FR regions that greatly employ sheet configuration. The HVRs in each chain are held together in close proximity by the FR region and together with the HVR from the other chain contribute to the formation of the antigen binding site of the anti-antigen binding site (Kabat et al., Sequences of (See Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Constant domains are not directly involved in antibody binding to antigen, but exhibit various effector functions such as the antibody's involvement in antibody-dependent cytotoxicity.

  The “light chain” of an antibody (immunoglobulin) from any mammalian species is one of two distinctly different forms called kappa (“κ”) and lambda (“λ”) based on the amino acid sequence of the constant domain. Can be assigned to one.

  As used herein, the term IgG “isotype” or “subclass” means any of the immunoglobulin subclasses defined by the chemical and antigenic properties of their constant regions.

Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are subclasses (isotypes), eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG _4. , IgA ₁ , and IgA ₂ can be further classified. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by a covalent or non-covalent association of the antibody with one or more other proteins or peptides.

  The terms “full-length antibody”, “intact antibody”, and “total antibody” are used interchangeably herein to refer to an antibody in its substantially intact form, not the antibody fragment described below. The These terms specifically refer to antibodies having a heavy chain that contains an Fc region.

  A “naked antibody” for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

“Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen-binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , and Fv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. It is done.

Two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and the remaining "Fc" fragments named reflecting their ability to crystallize easily by papain digestion of antibodies Is produced. Pepsin treatment yields an F (ab ′) ₂ fragment that has two antigen binding sites and is still capable of cross-linking antigen.

  “Fv” is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, the double chain Fv species consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In a single chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain associate in a “dimer” structure in which the light and heavy chains are similar to the structure in the double chain Fv species. As can be obtained, it can be covalently linked by a mobile peptide linker. It is in this configuration that the three HVRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen binding specificity to the antibody. However, the ability to recognize and bind to an antigen, even with a single variable domain (or half of an Fv containing only three HVRs specific for the antigen), with a lower affinity than the entire binding site. Have

The Fab fragment contains a heavy chain variable domain and a light chain variable domain, and also contains a light chain constant domain and a first heavy chain constant domain (CH1). Fab ′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain containing one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab ′ where the cysteine residue (s) of the constant domain have a free thiol group. F (ab ′) ₂ antibody fragments originally were produced as pairs of Fab ′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

  “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In general, scFv polypeptides further comprise a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For an overview of scFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. , (Springer-Verlag, New York, 1994), pp. 269-315.

  The term “diabody” refers to an antibody fragment having two antigen binding sites, which fragments are heavy chain variable domains (VH) (VH) connected to light chain variable domains (VL) within the same polypeptide chain. -VL). By using linkers that are too short to allow pairing between two domains on the same chain, these domains are paired with the complementary domains of another chain and the two antigen binding sites are separated. Make it. Diabodies can be bivalent or bispecific. For diabody, see, for example, EP 404,097, WO 1993/01161, Hudson et al. Nat. Med. 9: 129-134 (2003), and Hollinger et al. , Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9: 129-134 (2003).

  The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, eg, individual mutations that make up the population are possible mutations that may be present in small amounts. For example, except for naturally occurring mutations. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a target, wherein the target binding polypeptide sequence comprises a target binding polypeptide sequence from a plurality of polypeptide sequences. Obtained by a process involving selection. For example, the selection process can be the selection of a unique clone from multiple clones, such as a hybridoma clone, a phage clone, or a pool of recombinant DNA clones. The selected target binding sequence, for example, improves affinity for the target, humanizes the target binding sequence, improves its production in cell culture, reduces its immunogenicity in vivo, multispecific antibodies It should be understood that antibodies that may be further modified to produce and that include a modified target binding sequence are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed against a single determinant on the antigen. Oriented. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically not contaminated with other immunoglobulins.

  The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced, for example, using the hybridoma method (eg, Kohler and Milstein, Nature, 256: 495-97 (1975), Hongo et al., Hybridoma, 14 (3): 253-260 ( 1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hammerling et al., Monoclonal 68. 1981)), recombinant DNA methods (eg, US Pat. No. 4,816, 567), phage display technology (eg, Clackson et al., Nature, 352: 624-628 (1991), Marks et al., J. Mol. Biol. 222: 581-597 (1992)). Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004), Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004), and Lee et al., J. Immunol. Methods 284 (1-2): 119-132 (2004)), and Coordinate human immunoglobulin sequences For producing human or human-like antibodies in animals having human immunoglobulin loci or part or all of the genes (eg, WO1998 / 24893, WO1996 / 34096, WO1996 / 33735, WO1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993), Jakobovits et al., Nature 362: 255-258 (1993), Bruggemann et al., Year in Immunol.7: 33 (1993). 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661, 01 6, Marks et al. Bio / Technology 10: 779-783 (1992), Lonberg et al. , Nature 368: 856-859 (1994), Morrison, Nature 368: 812-813 (1994), Fishwild et al. , Nature Biotechnol. 14: 845-851 (1996), Neuberger, Nature Biotechnol. 14: 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)).

  Specifically, the monoclonal antibody in the present specification is an antibody in which a part of the heavy chain and / or light chain is derived from a specific species or an antibody belonging to a specific antibody class or subclass as long as it exhibits a desired biological activity. A “chimera” that is identical or homologous to the corresponding sequence in, while the remainder of the chain (s) is identical or homologous to the corresponding sequence in an antibody from another species or an antibody belonging to another antibody class or subclass Antibodies, as well as fragments of such antibodies (see, eg, US Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). . Chimeric antibodies include PRIMATTZED (registered trademark) antibodies, and the antigen-binding region of this antibody is derived, for example, from antibodies produced by immunization with macaque monkey antigens.

  “Humanized” forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, the humanized antibody is a non-human species, such as a mouse, rat, rabbit, or non-human primate, in which residues from the recipient's HVR have the desired specificity, affinity, and / or ability. It is a human immunoglobulin (recipient antibody) that is replaced by an HVR-derived residue of (donor antibody). In some examples, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the donor antibody. These modifications can be made to further refine antibody performance. In general, humanized antibodies comprise at least one, typically substantially all of the two variable domains, and all or substantially all of the hypervariable loops are hypervariable of non-human immunoglobulin. Corresponding to the loop, all or substantially all of the FRs are FRs of human immunoglobulin sequences. The humanized antibody optionally also includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see, for example, Jones et al. , Nature 321: 522-525 (1986), Riechmann et al. , Nature 332: 323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998), Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994) and U.S. Patent Nos. 6,982,321 and 7,087,409.

  A “human antibody” is an amino acid that is produced by a human and / or corresponds to the amino acid sequence of an antibody produced using any of the techniques for making a human antibody disclosed herein. An antibody having a sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, such as phage display libraries. Hoogenboom and Winter, J.A. Mol. Biol. , 227: 381 (1991), Marks et al. , J .; Mol. Biol. 222: 581 (1991). Cole et al. , Monoclonal Antibodies and Cancer Therapy, Alan R .; Liss, p. 77 (1985), Boerner et al. , J .; Immunol. 147 (1): 86-95 (1991) can also be used to prepare human monoclonal antibodies. van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001). Administering an antigen to a transgenic animal, eg, an immunized xenogeneic mouse, that has been modified to produce such an antibody in response to an antigen challenge, but whose endogenous locus has been abolished (See, for example, US Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE ™ technology). For human antibodies produced by human B cell hybridoma technology, see, for example, Li et al. , Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006).

A “species-dependent antibody” is an antibody that has a binding affinity for an antigen from a first mammalian species that is stronger than the binding affinity for a homologue of an antigen from a second mammalian species. Typically, species-dependent antibodies “bind specifically” to human antigens (eg, about 1 × 10 ⁻⁷ M or less, preferably about 1 × 10 ⁻⁸ M or less, preferably about 1 × 10 ⁻⁹ M Of an antigen from a second non-human mammalian species that has a binding affinity (Kd) value of at least about 50-fold, or at least about 500-fold, or at least about 1000-fold less than the binding affinity for a human antigen. Has binding affinity for homologues. The species-dependent antibody can be any of the various types of antibodies defined above, but is preferably a humanized antibody or a human antibody.

  As used herein, the terms “hypervariable region”, “HVR”, or “HV” refer to antibody variables whose sequence is hypervariable and / or forms a structurally defined loop. Refers to the domain area. In general, antibodies contain 6 HVRs, 3 at VH (H1, H2, H3) and 3 at VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the highest diversity of the six HVRs, and in particular H3 is thought to play a unique role in conferring superior specificity on the antibody. For example, Xu et al. , Immunity 13: 37-45 (2000), Johnson and Wu, Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Toyota, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al. , Nature 363: 446-448 (1993), Sheriff et al. , Nature Struct. Biol. 3: 733-736 (1996).

Several HVR depictions are used herein and are encompassed herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al., Sequences of Proteins of Immunological Institute, 5th Ed. Public Health Services, National Health Services, National Health, (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software. “Contact” HVR is based on analysis of available complex crystal structures. Residues from each of these HVRs are described below.

  HVRs may include the following “extended HVRs”: in VL, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), And in VH, 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). Variable domain residues are described in Kabat et al. Numbered according to (see above).

  “Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.

  The terms “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat” and variations thereof are described in Kabat et al. Refers to the numbering system used for the heavy chain variable domain or light chain variable domain of a compilation of antibodies in (see above). Using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to the FR or HVR truncation or insertion into the variable domain. For example, a heavy chain variable domain comprises a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and a residue inserted after heavy chain FR residue 82 (eg, a residue according to Kabat 82a, 82b, and 82c, etc.). Residue Kabat numbering can be determined for a given antibody by alignment of the sequence of the antibody with the sequence numbered by the “standard” Kabat.

  The Kabat numbering system is generally used when referring to residues in the variable domain (generally residues 1-107 of the light chain and residues 1-113 of the heavy chain) (see, eg, Kabat et al., Sequences of Immunological Inter. 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). “EU numbering system” or “EU index” is generally used when referring to residues in the immunoglobulin heavy chain constant region (eg, EU index reported in Kabat et al. (See above)). . “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.

  The expression “linear antibody” is described in Zapata et al. (1995 Protein Eng, 8 (10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

  As used herein, the terms “bind”, “specifically binds”, or “specifically” refer to the presence of a target in the presence of a heterogeneous population of molecules such as biological molecules. Refers to a measurable and reproducible interaction, such as binding between a target and an antibody. For example, an antibody that binds to, or specifically binds to, a target (which may be an epitope) is easier to bind to this target with higher affinity, binding power than it binds to other targets. And / or an antibody that binds for a longer period of time. In one embodiment, the degree of binding of the antibody to an irrelevant target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

  The term “detection” includes any detection means, including direct and indirect detection.

  As used herein, the term “biomarker” refers to an indicator that can be detected in a sample, eg, a predictive indicator, a diagnostic indicator, and / or a prognostic indicator. A biomarker may serve as an indicator of a particular subtype of a disease or disorder (eg, cancer) characterized by certain molecular, pathological, histological, and / or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include polynucleotides (eg, DNA and / or RNA), polynucleotide copy number alterations (eg, DNA copy number), polypeptides, polypeptides and polynucleotide modifications (eg, post-translational modifications), carbohydrates, and Examples include, but are not limited to, glycolipid-based molecular markers.

  The terms “biomarker signature”, “signature”, “biomarker expression signature”, or “expression signature” are used interchangeably herein to refer to a single biomarker or combination of biomarkers and their expression Is an indicator, eg, a predictive indicator, a diagnostic indicator, and / or a prognostic indicator. A biomarker signature can serve as an indicator of a particular subtype of a disease or disorder (eg, cancer) characterized by certain molecular, pathological, histological, and / or clinical features . In some embodiments, the biomarker signature is a “gene signature”. The term “gene signature” is used interchangeably with “gene expression signature” and refers to a polynucleotide or a combination of polynucleotides whose expression is indicative, eg, predictive indicator, diagnostic indicator, and / or prognosis. It is an indicator. In some embodiments, the biomarker signature is a “protein signature”. The term “protein signature” is used interchangeably with “protein expression signature” and refers to a single polypeptide or combination of polypeptides whose expression is indicative, eg, predictive indicator, diagnostic indicator, and / or prognosis. It is an indicator.

  The “amount” or “level” of a biomarker associated with an increased clinical benefit to an individual is a level that is detectable in a biological sample. These are known to those skilled in the art and can be measured by the methods disclosed herein. The expression level or amount of biomarker evaluated can be used to determine response to therapy.

  The terms “level of expression” or “expression level” are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. “Expression” generally refers to the process by which information (eg, genetic coding information and / or epigenetic information) is present in a cell and converted into a structure that operates there. Thus, as used herein, “expression” refers to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (eg, post-translational modification of a polypeptide). Can point to. Transcribed polynucleotides, translated polypeptides, or fragments of polynucleotides and / or polypeptide modifications (eg, post-translational modifications of polypeptides) are also transcripts produced or degraded by alternative splicing Regardless of whether it is derived from the transcript or from post-translational processing of the polypeptide, for example by proteolysis, it should be regarded as expressed. “Expressed genes” include genes that are transcribed as mRNA into a polynucleotide and then translated into a polypeptide, and also include genes that are transcribed into RNA but not translated into a polypeptide (eg, transfer RNA and ribosomal RNA). .

  “Elevated expression”, “increased expression level”, or “increased level” refers to an individual (s) not affected by a disease or disorder (eg, cancer) or an internal control (eg, a housekeeping biomarker) ) Refers to increased expression or increased level of the biomarker in an individual compared to a control such as

  A “reduced expression”, “reduced expression level”, or “reduced level” refers to an individual (s) not affected by a disease or disorder (eg, cancer) or an internal control (eg, a housekeeping biomarker). ) Refers to the reduced expression or reduced level of a biomarker in an individual compared to a control such as In some embodiments, reduced expression is little or no expression.

  The term “housekeeping biomarker” refers to a biomarker or group of biomarkers (eg, polynucleotides and / or polypeptides) that are typically present in all cell types as well. In some embodiments, the housekeeping biomarker is a “housekeeping gene”. A “housekeeping gene” as used herein refers to a gene or group of genes that encodes a protein whose activity is essential for the maintenance of cell function and is typically present in all cell types as well.

  As used herein, “amplification” generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” means at least two copies. “Copy” does not necessarily mean complete sequence complementarity or identity to the template sequence. For example, a copy can be a nucleotide analog such as deoxyinosine, an intentional sequence modification (such as a sequence modification introduced by a primer that can hybridize to a template but is not complementary), and / or during amplification. May contain sequence errors that occur in

  The term “multiplex PCR” refers to nucleic acids obtained from a single source (eg, an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction. Refers to the single PCR reaction performed above.

  The “stringency” of a hybridization reaction is readily determinable by one of ordinary skill in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, and shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, and lower temperatures are less likely. For further details and explanation of the stringency of hybridization reactions, see Ausubel et al. , Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

  “Stringent conditions” or “high stringency conditions” as defined herein are (1) those using low ionic strength and high temperature for washing, eg, 0.015 M sodium chloride / 0.0015 M Sodium citrate / 0.1% sodium dodecyl sulfate (50 ° C.), (2) using a denaturing agent such as formamide during hybridization, eg 50% (v / v) with 0.1% bovine serum albumin Formamide / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 750 mM sodium chloride, pH 6.5 50 mM sodium phosphate buffer (42 ° C.) with 75 mM sodium citrate, or (3) 0.2 X Wash for 10 minutes at 42 ° C in SSC (sodium chloride / sodium citrate), then 50% formamide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM phosphorous with 10 min high stringency wash consisting of 0.1 × SSC containing EDTA at 5 ° C. Use sodium acid (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate (42 ° C.) Can be identified by overnight hybridization in solution.

  “Moderately stringent conditions” are described in Sambrook et al. , Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, which can be specified as described in the use of wash solutions and hybridization conditions that are less stringent than those described above (eg, Temperature, ionic strength, and SDS%). Examples of moderately stringent conditions are 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% sulfuric acid. Overnight incubation at 37 ° C. in a solution containing dextran and 20 mg / mL denatured sheared salmon sperm DNA, followed by washing the filter in 1 × SSC at about 37-50 ° C. One skilled in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

  As used herein, the “polymerase chain reaction” or “PCR” technique generally refers to U.S. Pat. No. 4, published on Jul. 28, 1987, when a small amount of a specific piece of nucleic acid, RNA, and / or DNA is published. , 683,195, refers to a procedure that is amplified. In general, sequence information from the end of the region of interest or beyond must be available so that oligonucleotide primers can be designed, and the sequence of these primers is the opposite strand of the template to be amplified. Is the same or similar. The 5 'terminal nucleotides of these two primers can coincide with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, cDNA, bacteriophage sequences, plasmid sequences, etc. transcribed from total cellular RNA. See generally Mullis et al. , Cold Spring Harbor Symp. Quant. Biol. 51: 263 (1987), Erlich, ed. , PCR Technology, (Stockton Press, NY, 1989). As used herein, PCR is considered an example of a nucleic acid polymerase reaction method that amplifies a nucleic acid test sample, including the use of a known nucleic acid (DNA or RNA) as a primer, but the only example Rather, nucleic acid polymerases are utilized to amplify or generate specific pieces of nucleic acid, or to amplify or generate specific pieces of nucleic acid that are complementary to a specific nucleic acid.

  “Quantitative real-time polymerase chain reaction” or “qRT-PCR” refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique is described in Cronin et al. , Am. J. et al. Pathol. 164 (1): 35-42 (2004), and Ma et al. , Cancer Cell 5: 607-616 (2004).

  The term “microarray” refers to a regular arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.

  The term “polynucleotide”, when used in the singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. . Thus, for example, the polynucleotides defined herein include single-stranded and double-stranded DNA, single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and single-stranded And RNA comprising a double stranded region, a hybrid molecule comprising DNA and RNA, which may be single stranded or more typically double stranded, or may comprise single stranded and double stranded regions, It is not limited to these. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The chains within such regions can be from the same molecule or from different molecules. These regions may include all of one or more of these molecules, but more typically include only some regions of these molecules. One of the molecules in the triple helix region is often an oligonucleotide. The term “polynucleotide” specifically includes cDNA. The term includes DNA (including cDNA) and RNA containing one or more modified bases. Thus, DNA or RNA whose backbone has been modified for stability or for other reasons is a “polynucleotide” whose term is intended herein. Furthermore, DNA or RNA containing unusual bases such as inosine or modified bases such as tritiated bases are included in the term “polynucleotide” as defined herein. In general, the term “polynucleotide” refers to all chemically, enzymatically and / or metabolically modified forms of unmodified polynucleotides, as well as characteristics of viruses and cells (such as simple cells and complex cells). The chemical forms of DNA and RNA exhibiting

  The term “oligonucleotide” refers to a relatively short polynucleotide, including but not limited to single-stranded deoxyribonucleotides, single-stranded or double-stranded ribonucleotides, RNA: DNA hybrids, and double-stranded DNA. . Oligonucleotides such as single-stranded DNA probe oligonucleotides are often synthesized by chemical methods, for example, using a commercially available automated oligonucleotide synthesizer. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques, and by expression of DNA in cells and organisms.

  The term “diagnosis” as used herein is used herein to refer to the identification or classification of a molecular or pathological condition, disease, or condition (eg, cancer). For example, “diagnosis” may refer to the identification of a particular type of cancer. “Diagnosis” refers to a specific subtype (eg, a single biomarker or combination of biomarkers (eg, encoded by a particular gene or said gene), eg, by histopathological criteria or by molecular characteristics It may also refer to the subtype) cancer classification characterized by the expression of the protein).

  As used herein, the term “helping diagnosis” refers to a method that helps make a clinical determination regarding the presence or nature of a particular type of symptom or condition of a disease or disorder (eg, cancer). As used herein. For example, a method that aids in the diagnosis of a disease or condition (eg, cancer) can include measuring a particular biomarker in a biological sample from an individual.

  As used herein, the term “sample” refers to cells and / or other that are characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological properties. It refers to a composition obtained or derived from a target subject and / or individual that contains a molecular entity. For example, the phrase “disease sample” and variations thereof are obtained from a subject of interest that is expected or known to contain the cell and / or molecular entity being characterized. Refers to any given sample. Samples include primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells Urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof However, it is not limited to these.

  By “tissue sample” or “cell sample” is meant a collection of similar cells obtained from the tissue of a subject or individual. The tissue or cell sample source can be a fresh, frozen and / or stored organ, tissue sample, biopsy, and / or solid tissue from aspirate; blood or any blood component such as plasma; body fluid such as , Cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid; cells at any time during pregnancy or development of the subject. The tissue sample can also be a primary or cultured cell or cell line. Optionally, the tissue or cell sample is obtained from a diseased tissue / organ. Tissue samples can contain compounds that do not naturally mix with natural tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

  As used herein, a “reference sample”, “reference cell”, “reference tissue”, “control sample”, “control cell”, or “control tissue” is a sample used for comparison purposes, Refers to a cell, tissue, standard, or level. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is from a healthy and / or unaffected part (eg, tissue or cell) of the same subject or individual's body. can get. For example, healthy and / or unaffected cells or tissues adjacent to diseased cells or tissues (eg, cells or tissues adjacent to tumors). In another embodiment, the reference sample is obtained from untreated tissue and / or cells of the same subject or individual's body. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a healthy and / or unaffected portion of the body of an individual that is not the subject or individual (e.g., From tissue or cells). In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissue and / or cells of the body of an individual that is not the subject or individual.

  For purposes herein, a “section” of a tissue sample means a single portion or piece of a tissue sample, eg, a thin section of tissue or cells cut from a tissue sample. It is understood that multiple sections of a tissue sample can be taken and subjected to analysis, provided that the same section of tissue sample can be analyzed at both morphological and molecular levels, or polypeptide and poly Provided that it is understood that both nucleotides can be analyzed.

  “Correlating” or “correlating” means comparing the performance and / or results of the first analysis or protocol with the performance and / or results of the second analysis or protocol in any way. . For example, the results of the first analysis or protocol can be used in performing the second protocol and / or the results of the first analysis or protocol are used to perform the second analysis or protocol. You can decide what to do. With respect to polypeptide analysis or protocol embodiments, the results of polypeptide expression analysis or protocol can be used to determine if a particular treatment regimen should be performed. With respect to polynucleotide analysis or protocol embodiments, the results of polynucleotide expression analysis or protocol can be used to determine whether a particular treatment regimen should be performed.

  As used herein, the term “label” refers to a detectable compound or composition. The label is typically conjugated or fused directly or indirectly to a reagent such as a polynucleotide probe or antibody to facilitate detection of the reagent to which it is conjugated or fused. The label can itself be detectable (eg, a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze chemical modification of the substrate compound or composition resulting in a detectable product. .

  “Effective response” and similar wording of a patient or patient “responsiveness” to treatment with a medicament is clinically given to a patient at risk of or suffering from a disease or disorder such as cancer Refers to a therapeutic or therapeutic benefit. In one embodiment, such benefits may include prolonging survival (including overall survival and progression-free survival), providing an objective response (including complete or partial response), or a sign of cancer or Including any one or more of symptom improvements.

  A patient who “does not show an effective response” to treatment results in an extended survival (including overall survival and progression-free survival), an objective response (including complete or partial response), or Refers to patients who do not have any improvement in signs or symptoms of cancer.

  “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to Fc receptors (FcR) that are present on certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages) with secreted immunoglobulin. By binding, these cytotoxic effector cells refer to a form of cytotoxicity that allows them to specifically bind to target cells with antigen and then kill the target cells with cytotoxins. Monocytes express FcγRI, FcγRII, and FcγRIII, while NK cells, the primary cells that mediate ADCC, express FcγRIII only. FcR expression in hematopoietic cells is described in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), summarized in Table 3 of 464 Mitsugu. In order to evaluate the ADCC activity of the molecule of interest, the assay described in US Pat. No. 5,500,362 or US Pat. No. 5,821,337 or US Pat. No. 6,737,056 (Presta) In vitro ADCC assays can be performed. Useful effector cells for such assays include PBMC and NK cells. Alternatively or in addition, the ADCC activity of the molecule of interest can be determined in vivo, eg, in animal models such as Clynes et al. It can be evaluated in the animal model disclosed in PNAS (USA) 95: 652-656 (1998). An exemplary assay for assessing ADCC activity is provided in the Examples herein.

  “Complement dependent cytotoxicity” or “CDC” refers to lysis of a target cell in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass), which are bound to their cognate antigens. To assess complement activation, see, for example, Gazzano-Santoro et al. , J .; Immunol. The CDC assay described in Methods 202: 163 (1996) can be performed. For example, US Pat. No. 6,194,551 B1 and WO 1999/51642 for polypeptide variants having altered Fc region amino acid sequences (polypeptides having variant Fc regions) and for increasing or decreasing C1q binding ability. It is described in. For example, Idusogie et al. J. et al. Immunol. 164: 4178-4184 (2000).

  A “depleted anti-OX40 antibody” is an anti-OX40 antibody that kills or depletes OX40-expressing cells. Depletion of OX40 expressing cells can be achieved by various mechanisms such as antibody-dependent cell-mediated cytotoxicity and / or phagocytosis. Depletion of OX40 expressing cells can be assayed in vitro, and exemplary methods of in vitro ADCC and phagocytosis assays are provided herein. In some embodiments, the OX40-expressing cell is a human CD4 + effector T cell. In some embodiments, the OX40 expressing cells are transgenic BT474 cells that express human OX40.

  “Effector function” refers to biological activity attributable to the Fc region of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), phagocytosis, cell surface receptors (eg, B cell receptor Body) down-regulation and B cell activation.

  “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds to an IgG antibody (gamma receptor) and includes FcγRI, FcγRII, and FcγRIII subclass receptors, allelic variants and / or splice forms of those receptors including. FcγRII receptors include FcγRIIA (“activating receptors”) and FcγRIIB (“inhibiting receptors”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, eg, Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). For FcR, see, for example, Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), Capel et al. , Immunomethods 4: 25-34 (1994), and de Haas et al. , J .; Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including FcRs to be identified in the future, are encompassed by the term “FcR” herein. The terms “Fc receptor” or “FcR” refer to the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 ( 1994)) and the neonatal receptor FcRn involved in the control of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (eg, Ghetie and Ward., Immunol. Today 18 (12): 592-598 (1997), Ghetie et al., Nature Biotechnology, 15 (7): 637- 640 (1997), Hinton et al., J. Biol. Chem. 279 (8): 6213-6216 (2004), WO 2004/92219 (Hinton et al.) Binding to human FcRn in vivo. And the serum half-life of human FcRn high affinity binding polypeptides is, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which a polypeptide having a variant Fc region is administered. WO 2000/42072 (Presta) describes antibody variants with improved or reduced binding to FcR, eg Shields et al. J. Biol. Chem. 9 (2): 6591- See also 6604 (2001).

  A “functional Fc region” has an “effector function” of a native sequence Fc region. Exemplary “effector functions” include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, cell surface receptor downregulation (eg, B cell receptor, BCR), and the like. Such effector functions generally require binding of the Fc region to a binding domain (eg, an antibody variable domain) and can be assessed using, for example, various assays disclosed in the definitions herein.

  “Human effector cells” refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, these cells express at least FcγRIII and perform ADCC effector function (s). Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources such as blood.

  A cancer or biological sample “having human effector cells” is one that has human effector cells (eg, infiltrating human effector cells) present in the sample in a diagnostic test.

  A cancer or biological sample “having FcR-expressing cells” is one that has FcR expression (eg, infiltrating FcR-expressing cells) present in the sample in a diagnostic test. In some embodiments, the FcR is FcγR. In some embodiments, the FcR is an activated FcγR.

II. PD-1 Axis Binding Antagonist Provided herein is a method of treating cancer or delaying its progression in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist. Also provided herein is a method of enhancing immune function in an individual having cancer, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist.

  For example, PD-1 axis binding antagonists include PD-1 binding antagonists, PDL1 binding antagonists, and PDL2 binding antagonists. Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

  In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In one specific aspect, the ligand binding partner of PD-1 is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific embodiment, the binding partner of PDL1 is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In one specific aspect, the binding partner of PDL2 is PD-1. An antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

  In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), CT-011 (pizilizumab), MEDI-0680 (AMP-514). , PDR001, REGN2810, BGB-108, and BGB-A317. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (eg, an immunoad comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (eg, an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224, nivolumab, aka MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO ( (Registered trademark) is an anti-PD-1 antibody described in WO 2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA (registered trademark), and SCH-900475, are described in WO 2009/114335. Anti-PD-1 CT-011, aka hBAT, hBAT-1, or pilizizumab is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, aka B7-DCIg is WO2010 / 027827 and WO2011. PDL2-Fc fusion soluble receptor described in / 066342.

（配列番号１０）、または
（ｂ）軽鎖配列は、以下の軽鎖配列と少なくとも８５％、少なくとも９０％、少なくとも９１％、少なくとも９２％、少なくとも９３％、少なくとも９４％、少なくとも９５％、少なくとも９６％、少なくとも９７％、少なくとも９８％、少なくとも９９％、または１００％の配列同一性を有する：

（配列番号１１）。 In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number 946414-94-4). In yet a further embodiment, an isolated anti-PD comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 10 and / or a light chain variable region comprising the light chain variable region amino acid sequence of SEQ ID NO: 11. -1 antibody is provided. In yet a further embodiment, an isolated anti-PD-1 antibody comprising a heavy chain sequence and / or a light chain sequence is provided,
(A) the heavy chain sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% with the following heavy chain sequence: Have at least 98%, at least 99%, or 100% sequence identity:

(SEQ ID NO: 10), or (b) the light chain sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least Has sequence identity of 96%, at least 97%, at least 98%, at least 99%, or 100%:

(SEQ ID NO: 11).

（配列番号１２）、または
（ｂ）軽鎖配列は、以下の軽鎖配列と少なくとも８５％、少なくとも９０％、少なくとも９１％、少なくとも９２％、少なくとも９３％、少なくとも９４％、少なくとも９５％、少なくとも９６％、少なくとも９７％、少なくとも９８％、少なくとも９９％、または１００％の配列同一性を有する：

（配列番号１３）。 In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS registry number 1374853-91-4). In yet a further embodiment, an isolated anti-PD comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 12 and / or a light chain variable region comprising the light chain variable region amino acid sequence of SEQ ID NO: 13. -1 antibody is provided. In yet a further embodiment, an isolated anti-PD-1 antibody comprising a heavy chain sequence and / or a light chain sequence is provided,
(A) the heavy chain sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% with the following heavy chain sequence: Have at least 98%, at least 99%, or 100% sequence identity:

(SEQ ID NO: 12), or (b) the light chain sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least Has sequence identity of 96%, at least 97%, at least 98%, at least 99%, or 100%:

(SEQ ID NO: 13).

  In some embodiments, the PDL1 binding antagonist is an anti-PDL1 antibody. In some embodiments, the PDL1 binding antagonist is YW243.55. It is selected from the group consisting of S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avermelb). MDX-1105, also known as BMS-936559, is an anti-PDL1 antibody described in WO2007 / 005874. Antibody YW243.55. S70 (heavy chain variable region sequence and light chain variable region sequence shown in SEQ ID NOs: 20 and 21, respectively) is anti-PDL1 described in WO2010 / 077634 A1. MEDI4736 is an anti-PDL1 antibody described in WO2011 / 066389 and US2013 / 034559.

  Examples of anti-PDL1 antibodies useful in the methods of the present invention and methods of making them are described in PCT patent application WO2010 / 077634 A1 and US Pat. No. 8,217,149, which are hereby incorporated by reference. Incorporated.

In some embodiments, the PD-1 axis binding antagonist is an anti-PDL1 antibody. In some embodiments, the anti-PDL1 antibody can inhibit binding between PDL1 and PD-1 and / or binding between PDL1 and B7-1. In some embodiments, the anti-PDL1 antibody is a monoclonal antibody. In some embodiments, the anti-PDL1 antibody is an antibody fragment selected from the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab ′) ₂ fragments. In some embodiments, the anti-PDL1 antibody is a humanized antibody. In some embodiments, the anti-PDL1 antibody is a human antibody.

  Anti-PDL1 antibodies useful in the present invention, including compositions containing such antibodies, such as those described in WO2010 / 077634 A1, can be used in combination with OX40 binding agonists to treat cancer. In some embodiments, the anti-PDL1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9.

さらに、Ｘ_１は、ＤまたはＧであり、Ｘ_２は、ＳまたはＬであり、Ｘ_３は、ＴまたはＳである。 In one embodiment, the anti-PDL1 antibody contains a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences;

Further, X ₁ is D or G, X ₂ is S or L, and X ₃ is T or S.

In one specific aspect, X ₁ is D, X ₂ is S, and X ₃ is T. In another aspect, the polypeptide further comprises a variable region heavy chain framework sequence juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR) -H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In a further aspect, the framework sequence is a VH subgroup III consensus framework. In yet a further aspect, at least one of the framework sequences is:

さらに、Ｘ_４は、ＤまたはＶであり、Ｘ_５は、ＶまたはＩであり、Ｘ_６は、ＳまたはＮであり、Ｘ_７は、ＡまたはＦであり、Ｘ_８は、ＶまたはＬであり、Ｘ_９は、ＦまたはＴであり、Ｘ_１０は、ＹまたはＡであり、Ｘ_１１は、Ｙ、Ｇ、Ｆ、またはＳであり、Ｘ_１２は、Ｌ、Ｙ、Ｆ、またはＷであり、Ｘ_１３は、Ｙ、Ｎ、Ａ、Ｔ、Ｇ、Ｆ、またはＩであり、Ｘ_１４は、Ｈ、Ｖ、Ｐ、Ｔ、またはＩであり、Ｘ_１５は、Ａ、Ｗ、Ｒ、Ｐ、またはＴである。 In yet a further aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising HVR-L1, HVR-L2, and HVR-L3,

Further, X ₄ is D or V, X ₅ is V or I, X ₆ is S or N, X ₇ is A or F, and X ₈ is V or L. X ₉ is F or T, X ₁₀ is Y or A, X ₁₁ is Y, G, F, or S, and X ₁₂ is L, Y, F, or W X ₁₃ is Y, N, A, T, G, F, or I, X ₁₄ is H, V, P, T, or I, and X ₁₅ is A, W, R, P or T.

In yet a further aspect, X ₄ is D, X ₅ is V, X ₆ is S, X ₇ is A, X ₈ is V, and X ₉ is F, X ₁₀ is Y, X ₁₁ is Y, X ₁₂ is L, X ₁₃ is Y, X ₁₄ is H, and X ₁₅ is A. is there. In yet a further aspect, the light chain further comprises a variable region light chain framework sequence juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-( HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet a further aspect, the framework sequence is derived from a human consensus framework sequence. In yet a further aspect, the framework sequence is a VL kappa I consensus framework. In yet a further aspect, at least one of the framework sequences is:

さらに、Ｘ_１は、ＤまたはＧであり、Ｘ_２は、ＳまたはＬであり、Ｘ_３は、ＴまたはＳであり、Ｘ_４は、ＤまたはＶであり、Ｘ_５は、ＶまたはＩであり、Ｘ_６は、ＳまたはＮであり、Ｘ_７は、ＡまたはＦであり、Ｘ_８は、ＶまたはＬであり、Ｘ_９は、ＦまたはＴであり、Ｘ_１０は、ＹまたはＡであり、Ｘ_１１は、Ｙ、Ｇ、Ｆ、またはＳであり、Ｘ_１２は、Ｌ、Ｙ、Ｆ、またはＷであり、Ｘ_１３は、Ｙ、Ｎ、Ａ、Ｔ、Ｇ、Ｆ、またはＩであり、Ｘ_１４は、Ｈ、Ｖ、Ｐ、Ｔ、またはＩであり、Ｘ_１５は、Ａ、Ｗ、Ｒ、Ｐ、またはＴである。 In another embodiment, an isolated anti-PDL1 antibody or antigen-binding fragment comprising a heavy chain variable region sequence and a light chain variable region sequence is provided,

Further, X ₁ is D or G, X ₂ is S or L, X ₃ is T or S, X ₄ is D or V, and X ₅ is V or I. X ₆ is S or N, X ₇ is A or F, X ₈ is V or L, X ₉ is F or T, X ₁₀ is Y or A Yes, X ₁₁ is Y, G, F, or S, X ₁₂ is L, Y, F, or W, and X ₁₃ is Y, N, A, T, G, F, or I X ₁₄ is H, V, P, T, or I, and X ₁₅ is A, W, R, P, or T.

In one specific aspect, X ₁ is D, X ₂ is S, and X ₃ is T. In another aspect, X ₄ is D, X ₅ is V, X ₆ is S, X ₇ is A, X ₈ is V, and X ₉ is F And X ₁₀ is Y, X ₁₁ is Y, X ₁₂ is L, X ₁₃ is Y, X ₁₄ is H, and X ₁₅ is A. . In yet another aspect, X ₁ is D, X ₂ is S, X ₃ is T, X ₄ is D, X ₅ is V, and X ₆ is S, X ₇ is A, X ₈ is V, X ₉ is F, X ₁₀ is Y, X ₁₁ is Y, X ₁₂ is L Yes, X ₁₃ is Y, X ₁₄ is H, and X ₁₅ is A.

In a further aspect, the heavy chain variable region comprises (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC- FR4) contains one or more framework sequences juxtaposed between HVRs, the light chain variable region is (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2) Includes one or more framework sequences juxtaposed between HVRs, such as-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet a further aspect, the framework sequence is derived from a human consensus framework sequence. In yet a further aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet a further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further aspect, one or more of the heavy chain framework sequences are:

In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet a further aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet a further aspect, one or more of the light chain framework sequences are:

  In yet a more specific aspect, the antibody further comprises a human or mouse constant region. In yet a further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet a more specific aspect, the human constant region is IgG1. In yet a further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet a further aspect, the mouse constant region is IgG2A. In yet a more specific aspect, the antibody has reduced or minimal effector function. In an even more specific aspect, the minimal effector function is due to “effectorless Fc mutation” or aglycosylation. In yet a further embodiment, the effectorless Fc mutation is a N297A or D265A / N297A substitution in the constant region.

In yet another embodiment, an anti-PDL1 antibody comprising a heavy chain variable region sequence and a light chain variable region sequence is provided,
(A) The heavy chains are HVR-H1, HVR-H2, and HVR having at least 85% sequence identity with GFTFSDSWIH (SEQ ID NO: 1), AWISPYGGSTYADSVKG (SEQ ID NO: 2), and RHWPCGGFDY (SEQ ID NO: 3), respectively. -B further comprising a H3 sequence, or Further included are L1, HVR-L2, and HVR-L3 sequences.

In one specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100%. In another aspect, the heavy chain variable region is (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC- FR4) contains one or more framework sequences juxtaposed between HVRs, the light chain variable region is (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2) Includes one or more framework sequences juxtaposed between HVRs, such as-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet a further aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet a further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further aspect, one or more of the heavy chain framework sequences are:

In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet a further aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet a further aspect, one or more of the light chain framework sequences are:

  In yet a more specific aspect, the antibody further comprises a human or mouse constant region. In yet a further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet a more specific aspect, the human constant region is IgG1. In yet a further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet a further aspect, the mouse constant region is IgG2A. In yet a more specific aspect, the antibody has reduced or minimal effector function. In an even more specific aspect, the minimal effector function is due to “effectorless Fc mutation” or aglycosylation. In yet a further embodiment, the effectorless Fc mutation is a N297A or D265A / N297A substitution in the constant region.

（配列番号２８）と少なくとも８５％の配列同一性を有し、または
（ｂ）軽鎖配列は、軽鎖配列

（配列番号９）と少なくとも８５％の配列同一性を有する。 In yet a further embodiment, an isolated anti-PDL1 antibody comprising a heavy chain variable region sequence and a light chain variable region sequence is provided,
(A) The heavy chain sequence is a heavy chain sequence

(SEQ ID NO: 28) has at least 85% sequence identity, or (b) the light chain sequence is a light chain sequence

(SEQ ID NO: 9) has at least 85% sequence identity.

In one specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100%. In another aspect, the heavy chain variable region is (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC- FR4) contains one or more framework sequences juxtaposed between HVRs, the light chain variable region is (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2) Includes one or more framework sequences juxtaposed between HVRs, such as-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In a further aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet a further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further aspect, one or more of the heavy chain framework sequences are:

In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet a further aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet a further aspect, one or more of the light chain framework sequences are:

  In yet a more specific aspect, the antibody further comprises a human or mouse constant region. In yet a further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet a more specific aspect, the human constant region is IgG1. In yet a further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet a further aspect, the mouse constant region is IgG2A. In yet a more specific aspect, the antibody has reduced or minimal effector function. In yet a more specific embodiment, the minimal effector function is due to production in prokaryotic cells. In an even more specific aspect, the minimal effector function is due to “effectorless Fc mutation” or aglycosylation. In yet a further embodiment, the effectorless Fc mutation is a N297A or D265A / N297A substitution in the constant region.

In another further embodiment, an isolated anti-PDL1 antibody comprising a heavy chain variable region sequence and a light chain variable region sequence is provided,
(A) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence IbuikyuerubuiiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesudiesudaburyuaieichidaburyubuiarukyueiPijikeijieruidaburyubuieidaburyuaiesuPiwaijijiesutiwaiwaieidiesubuikeijiaruefutiaiesueiditiesukeienutieiwaierukyuemuenuesueruarueiiditieibuiYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7), or (b) the light chain sequence, a light chain sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9) Have at least 85% sequence identity.

（配列番号８）と少なくとも８５％の配列同一性を有し、または
（ｂ）軽鎖配列は、軽鎖配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＲＡＳＱＤＶＳＴＡＶＡＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＳＡＳＦ ＬＹＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＦＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＹＬＹＨＰＡＴＦＧＱＧＴＫＶＥＩＫＲ（配列番号９）と少なくとも８５％の配列同一性を有する。 In yet a further embodiment, an isolated anti-PDL1 antibody comprising a heavy chain variable region sequence and a light chain variable region sequence is provided,
(A) The heavy chain sequence is a heavy chain sequence

(B) the light chain sequence has the light chain sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASF LYSGVPSRFSGSGGTDFTLTISLQPEDFATYCQFLYQ

In one specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100%. In another aspect, the heavy chain variable region is (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC- FR4) contains one or more framework sequences juxtaposed between HVRs, the light chain variable region is (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2) Includes one or more framework sequences juxtaposed between HVRs, such as-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In a further aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet a further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further aspect, one or more of the heavy chain framework sequences are:

In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet a further aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet a further aspect, one or more of the light chain framework sequences are:

  In yet a more specific aspect, the antibody further comprises a human or mouse constant region. In yet a further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet a more specific aspect, the human constant region is IgG1. In yet a further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet a further aspect, the mouse constant region is IgG2A. In yet a more specific aspect, the antibody has reduced or minimal effector function. In yet a more specific embodiment, the minimal effector function is due to production in prokaryotic cells. In an even more specific aspect, the minimal effector function is due to “effectorless Fc mutation” or aglycosylation. In yet a further embodiment, the effectorless Fc mutation is a N297A or D265A / N297A substitution in the constant region.

（配列番号２９）、及び／または
（ｂ）軽鎖配列は、以下の軽鎖配列と少なくとも８５％、少なくとも９０％、少なくとも９１％、少なくとも９２％、少なくとも９３％、少なくとも９４％、少なくとも９５％、少なくとも９６％、少なくとも９７％、少なくとも９８％、少なくとも９９％、または１００％の配列同一性を有する：

（配列番号３０）。 In yet another embodiment, the anti-PDL1 antibody is MPDL3280A (CAS Registry Number 1422185-06-5). In still a further embodiment, the heavy chain variable region amino acid sequence IbuikyuerubuiiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesudiesudaburyuaieichidaburyubuiarukyueiPijikeijieruidaburyubuieidaburyuaiesuPiwaijijiesutiwaiwaieidiesubuikeijiaruefutiaiesueiditiesukeienutieiwaierukyuemuenuesueruarueiiditieibuiYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK heavy chain variable region (SEQ ID NO: 8), and the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTA Provided is an isolated anti-PDL1 antibody comprising a light chain variable region comprising VAWYQQKPGKAPKLLIY SASF LYSGVPSFSGGSGSDFLTTISLSQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9). In yet a further embodiment, an isolated anti-PDL1 antibody comprising a heavy chain sequence and / or a light chain sequence is provided,
(A) the heavy chain sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% with the following heavy chain sequence: Have at least 98%, at least 99%, or 100% sequence identity:

(SEQ ID NO: 29), and / or (b) the light chain sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% with the following light chain sequence: Having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity:

(SEQ ID NO: 30).

  In yet a further embodiment, the invention provides a composition comprising any of the above-described anti-PDL1 antibodies in combination with at least one pharmaceutically acceptable carrier.

In yet a further embodiment, an isolated nucleic acid encoding a light chain variable region sequence or heavy chain variable region sequence of an anti-PDL1 antibody is provided,
(A) The heavy chains are HVR-H1, HVR-H2, and HVR having at least 85% sequence identity with GFTFSDSWIH (SEQ ID NO: 1), AWISPYGGSTYADSVKG (SEQ ID NO: 2), and RHWPCGGFDY (SEQ ID NO: 3), respectively. -Further comprising an H3 sequence;
(B) The light chains are HVR-L1, HVR-L2, and HVR having at least 85% sequence identity with RASQDVSTAVA (SEQ ID NO: 4), SASFLYS (SEQ ID NO: 5), and QQYLYHPAT (SEQ ID NO: 6), respectively. -Further comprises the L3 sequence.

In one specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100%. In an aspect, the heavy chain variable region comprises (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4) And the light chain variable region comprises (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-( It includes one or more framework sequences juxtaposed between HVRs such as LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In a further aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet a further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet a further aspect, one or more of the heavy chain framework sequences are:

In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet a further aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet a further aspect, one or more of the light chain framework sequences are:

  In yet a more specific aspect, the antibodies described herein (such as anti-PD-1 antibody, anti-PDL1 antibody, or anti-PDL2 antibody) further comprise a human or mouse constant region. In yet a further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet a more specific aspect, the human constant region is IgG1. In yet a further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet a further aspect, the mouse constant region is IgG2A. In yet a more specific aspect, the antibody has reduced or minimal effector function. In yet a more specific embodiment, the minimal effector function is due to production in prokaryotic cells. In an even more specific aspect, the minimal effector function is due to “effectorless Fc mutation” or aglycosylation. In yet a further aspect, the Fc mutation without effector is a N297A or D265A / N297A substitution within the constant region.

  In yet a further aspect, provided herein is a nucleic acid encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expression of a nucleic acid encoding any of the aforementioned anti-PDL1, anti-PD-1, or anti-PDL2 antibodies. In yet a more specific aspect, the vector further comprises a host cell suitable for nucleic acid expression. In yet a more specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In yet a more specific embodiment, the eukaryotic cell is a mammalian cell such as a Chinese hamster ovary (CHO).

  The antibody or antigen-binding fragment thereof encodes, for example, any of the aforementioned anti-PDL1, anti-PD-1, or anti-PDL2 antibodies, or antigen-binding fragments using methods known in the art. It can be made by a process comprising culturing host cells containing the nucleic acid in a form suitable for expression under conditions suitable for the production of such antibodies or fragments and recovering the antibodies or fragments.

  In some embodiments, the isolated anti-PDL1 antibody is aglycosylated. Antibody glycosylation is typically either N-linked or O-linked. N-linked refers to the binding of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for enzyme binding to the asparagine side chain of the carbohydrate moiety. Thus, the presence of any of these tripeptide sequences within the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to binding to one hydroxy amino acid of sugars, N-acetylgalactosamine, galactose, or xylose, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxy Lysine can also be used. Removal of the glycosylation site from the antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-described tripeptide sequences is removed (for N-linked glycosylation sites). This modification can be done by replacing an asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (eg, glycine, alanine or a conservative substitution).

  In any of the embodiments herein, the isolated anti-PDL1 antibody binds to human PDL1, eg, human PDL1 as shown in UniProtKB / Swiss-Prot accession number Q9NZQ7.1, or a variant thereof. be able to.

  In yet a further embodiment, the present invention comprises an anti-PDL1, anti-PD-1, or anti-PDL2 antibody provided herein, or an antigen-binding fragment thereof, and at least one pharmaceutically acceptable carrier. A composition comprising is provided. In some embodiments, an anti-PDL1, anti-PD-1, or anti-PDL2 antibody, or antigen-binding fragment thereof administered to an individual is a composition comprising one or more pharmaceutically acceptable carriers. Any of the pharmaceutically acceptable carriers described herein or known in the art can be used.

  In some embodiments, an anti-PDL1 antibody described herein has an amount of the antibody of about 60 mg / mL, histidine acetate at a concentration of about 20 mM, sucrose at a concentration of about 120 mM, and 0.04% (w Present in a formulation comprising a polysorbate at a concentration of / v) (eg, polysorbate 20), which has a pH of about 5.8. In some embodiments, an anti-PDL1 antibody described herein has an amount of the antibody of about 125 mg / mL, histidine acetate at a concentration of about 20 mM, sucrose at a concentration of about 240 mM, and 0.02% (w Present in a formulation comprising a polysorbate at a concentration of / v) (eg, polysorbate 20), which formulation has a pH of about 5.5.

III. OX40 Binding Agonists Provided herein are methods for treating cancer or delaying its progression in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist. Also provided herein is a method of enhancing immune function in an individual having cancer, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist.

  Examples of the OX40 binding agonist include OX40 agonist antibody (for example, anti-human OX40 agonist antibody), OX40L agonist fragment, OX40 oligomer receptor, and OX40 immunoadhesin.

  In some embodiments, the OX40 agonist antibody increases CD4 + effector T cell proliferation and / or cytokine production by CD4 + effector T cells compared to pre-treatment proliferation and / or cytokine production with an OX40 agonist antibody. Increase. In some embodiments, the cytokine is IFN-γ.

  In some embodiments, the OX40 agonist antibody increases memory T cell proliferation and / or increases cytokine production by memory cells. In some embodiments, the cytokine is IFN-γ.

  In some embodiments, the OX40 agonist antibody inhibits Treg suppression of effector T cell function. In some embodiments, the effector T cell function is effector T cell proliferation and / or cytokine production. In some embodiments, the effector T cell is a CD4 + effector T cell.

  In some embodiments, the OX40 agonist antibody increases OX40 signaling in target cells that express OX40. In some embodiments, OX40 signaling is detected by monitoring NFkB downstream signaling.

  In some embodiments, the anti-human OX40 agonist antibody is a depleted anti-human OX40 antibody (eg, depletes cells that express human OX40). In some embodiments, the human OX40 expressing cells are CD4 + effector T cells. In some embodiments, the human OX40 expressing cell is a Treg cell. In some embodiments, the depletion is due to ADCC and / or phagocytosis. In some embodiments, the antibody mediates ADCC by binding FcγR expressed by human effector cells and activating human effector cell function. In some embodiments, the antibody mediates phagocytosis by binding FcγR expressed by human effector cells and activating human effector cell function. Illustrative human effector cells include, for example, macrophages, natural killer (NK) cells, monocytes, and neutrophils. In some embodiments, the human effector cell is a macrophage.

  In some embodiments, the anti-human OX40 agonist antibody has a functional Fc region. In some embodiments, the effector function of the functional Fc region is ADCC. In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region is human IgG4.

  In some embodiments, the anti-human OX40 agonist antibody is a human antibody or a humanized antibody. In some embodiments, the OX40 binding agonist (eg, OX40 agonist antibody) is not MEDI6383. In some embodiments, the OX40 binding agonist (eg, OX40 agonist antibody) is not MEDI0562.

（配列番号３１）を含む重鎖及び／または配列

（配列番号３２）を含む軽鎖を含む。いくつかの実施形態では、本抗体は、米国特許第７，５５０，１４０号に記載の抗体００８の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、米国特許第７，５５０，１４０号に記載の抗体００８の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in US Pat. No. 7,550,140, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain and / or sequence comprising (SEQ ID NO: 31)

A light chain comprising (SEQ ID NO: 32). In some embodiments, the antibody is at least one, two, three, four, five, or six hypervariable regions (HVRs) of antibody 008 described in US Pat. No. 7,550,140. ) Contains the sequence. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody 008 described in US Pat. No. 7,550,140.

（配列番号３３）の配列を含む。いくつかの実施形態では、本抗体は、米国特許第７，５５０，１４０号に記載の抗体ＳＣ０２００８の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、米国特許第７，５５０，１４０号に記載の抗体ＳＣ０２００８の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in US Pat. No. 7,550,140. In some embodiments, the anti-human OX40 agonist antibody is

The sequence of (SEQ ID NO: 33) is included. In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable regions (HVRs) of antibody SC02008 as described in US Pat. No. 7,550,140. ) Contains the sequence. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody SC02008 as described in US Pat. No. 7,550,140.

（配列番号３４）を含む重鎖及び／または配列

（配列番号３５）を含む軽鎖を含む。いくつかの実施形態では、本抗体は、米国特許第７，５５０，１４０号に記載の抗体０２３の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、米国特許第７，５５０，１４０号に記載の抗体０２３の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in US Pat. No. 7,550,140. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain and / or sequence comprising (SEQ ID NO: 34)

A light chain comprising (SEQ ID NO: 35). In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable regions (HVRs) of antibody 023 as described in US Pat. No. 7,550,140. ) Contains the sequence. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody 023, as described in US Pat. No. 7,550,140.

  In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in US Pat. No. 7,960,515, which is hereby incorporated by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody comprises a light chain variable region comprising the heavy chain variable region and / or sequences DiaikyuemutikyuesuPiesuesueruesueiesubuijidiarubuitiaitishiarueiesukyujiaiesuesudaburyuerueidaburyuwaikyukyukeiPiikeieiPikeiesueruaiwaieieiesuesuerukyuesujibuiPiesuaruefuesujiesujiesujitidiefutierutiaiesuesuerukyuPiidiFATYYCQQYNSYPPTFGGGTKVEIK (SEQ ID NO: 37) comprising the sequence IbuikyuerubuiiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesuesuwaiesuemuenudaburyubuiarukyueiPijikeijieruidaburyubuiesuwaiaiesuesuesuesuesutiaidiwaieidiesubuikeijiaruefutiaiesuarudienueikeienuesueruwaierukyuemuenuesueruarudiiditieibuiYYCARESGWYLFDYWGQGTLVTVSS (SEQ ID NO: 36). In some embodiments, the antibody is at least one, two, three, four, five, or six hypervariable regions (HVRs) of antibody 11D4 as described in US Pat. No. 7,960,515. ) Contains the sequence. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or light chain variable region sequence of antibody 11D4 as described in US Pat. No. 7,960,515.

（配列番号３８）を含む重鎖可変領域及び／または配列ＥＩＶＶＴＱＳＰＡＴＬＳＬＳＰＧＥＲＡＴＬＳＣＲＡＳＱＳＶＳＳＹＬＡＷＹＱＱＫＰＧＱＡＰＲＬＬＩＹＤＡＳＮＲＡＴＧＩＰＡＲＦＳＧＳＧＳＧＴＤＦＴＬＴＩＳＳＬＥＰＥＤＦＡＶＹＹＣＱＱＲＳＮＷＰＴＦＧＱＧＴＫＶＥＩＫ（配列番号３９）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、米国特許第７，９６０，５１５号に記載の抗体１８Ｄ８の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、米国特許第７，９６０，５１５号に記載の抗体１８Ｄ８の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in US Pat. No. 7,960,515. In some embodiments, the anti-human OX40 agonist antibody has a sequence

A heavy chain variable region comprising (SEQ ID NO: 38) and / or a sequence comprising EIVVTQSPATLSLPSGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGGSGTDFLTTISLEPTEDFAVYYCQRSNWPPTFGQGTKVEIK (variable light number region 39). In some embodiments, the antibody is at least one, two, three, four, five, or six hypervariable regions (HVRs) of antibody 18D8 described in US Pat. No. 7,960,515. ) Contains the sequence. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody 18D8 described in US Pat. No. 7,960,515.

（配列番号４０）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＫＡＳＱＤＶＳＴＡＶＡＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＳＡＳＹＬＹＴＧＶＰＳＲＦＳＧＳＧＳＧＴＤＦＴＦＴＩＳＳＬＱＰＥＤＩＡＴＹＹＣＱＱＨＹＳＴＰＲＴＦＧＱＧＴＫＬＥＩＫ（配列番号４１）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１２／０２７３２８に記載の抗体ｈｕ１０６−２２２の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１２／０２７３２８に記載の抗体ｈｕ１０６−２２２の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2012 / 027328, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 40) and / or the sequence DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYLYTGVPSFSGSGSGDFFTISSLQPEDIATYCQQHYSTPRTFFGQGTKLEIK In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody hu106-222 as described in WO2012 / 027328. . In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody hu106-222, as described in WO2012 / 027328.

（配列番号４２）を含む重鎖可変領域及び／または配列ＥＩＶＬＴＱＳＰＡＴＬＳＬＳＰＧＥＲＡＴＬＳＣＲＡＳＫＳＶＳＴＳＧＹＳＹＭＨＷＹＱＱＫＰＧＱＡＰＲＬＬＩＹＬＡＳＮＬＥＳＧＶＰＡＲＦＳＧＳＧＳＧＴＤＦＴＬＴＩＳＳＬＥＰＥＤＦＡＶＹＹＣＱＨＳＲＥＬＰＬＴＦＧＧＧＴＫＶＥＩＫ（配列番号４３）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１２／０２７３２８に記載の抗体Ｈｕ１１９−１２２の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１２／０２７３２８に記載の抗体Ｈｕ１１９−１２２の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2012 / 027328. In some embodiments, the anti-human OX40 agonist antibody has a sequence

A heavy chain variable region comprising (SEQ ID NO: 42) and / or a sequence EIVLTQSPATLSLPSGERATLSCRASKSVSTSSGYSYMHWYQQKPGQAPRLLILYLASNLESGVPARFSSGSGTFDFLTISSLEPEDFAVYYQQHSLRLTPLTGGGGTKVEIK In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody Hu119-122 described in WO2012 / 027328. . In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody Hu119-122 as described in WO2012 / 027328.

（配列番号４４）を含む重鎖及び／または配列

（配列番号４５）を含む軽鎖を含む。いくつかの実施形態では、抗ヒトＯＸ４０アゴニスト抗体は、配列

（配列番号６１）を含む重鎖可変領域及び／または配列

（配列番号６２）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１３／０２８２３１に記載の抗体Ｍａｂ ＣＨ １１９−４３−１の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１３／０２８２３１に記載の抗体Ｍａｂ ＣＨ １１９−４３−１の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2013 / 028231, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain and / or sequence comprising (SEQ ID NO: 44)

A light chain comprising (SEQ ID NO: 45). In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region and / or sequence comprising (SEQ ID NO: 61)

A light chain variable region comprising (SEQ ID NO: 62). In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable regions (HVRs) of antibody Mab CH 119-43-1 as described in WO2013 / 028231. ) Contains the sequence. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody Mab CH 119-43-1 as described in WO2013 / 028231.

（配列番号４６）を含む重鎖可変領域及び／または配列

（配列番号４７）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１３／０３８１９１に記載の抗体クローン２０Ｅ５の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１３／０３８１９１に記載の抗体クローン２０Ｅ５の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is the anti-human OX40 agonist antibody described in WO2013 / 038191, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region and / or sequence comprising (SEQ ID NO: 46)

A light chain variable region comprising (SEQ ID NO: 47). In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 as described in WO2013 / 038191. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody clone 20E5 as described in WO2013 / 038191.

（配列番号４８）を含む重鎖可変領域及び／または配列ＤＩＶＭＴＱＳＨＫＦＭＳＴＳＬＧＤＲＶＳＩＴＣＫＡＳＱＤＶＧＡＡＶＡＷＹＱＱＫＰＧＱＳＰＫＬＬＩＹＷＡＳＴＲＨＴＧＶＰＤＲＦＴＧＧＧＳＧＴＤＦＴＬＴＩＳＮＶＱＳＥＤＬＴＤＹＦＣＱＱＹＩＮＹＰＬＴＦＧＧＧＴＫＬＥＩＫＲ（配列番号４９）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１３／０３８１９１に記載の抗体クローン１２Ｈ３の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１３／０３８１９１に記載の抗体クローン１２Ｈ３の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO2013 / 038191. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 48) and / or the sequence DIVMTQSHKMSTSLGDRVSITCKASQDVGAAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGGGSGTDFLTTISNVQDLTDYFCQQYINLYPLTFGGGTKL In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2013 / 038191. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody clone 12H3 as described in WO2013 / 038191.

（配列番号５０）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＲＡＳＱＤＩＳＮＹＬＮＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＹＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＧＮＴＬＰＷＴＦＧＱＧＴＫＶＥＩＫＲ（配列番号５１）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is the anti-human OX40 agonist antibody described in WO2014 / 148895A1, which is incorporated herein by reference in its entirety. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 50) and / or the sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLYNWYQQKPGKAPKLLIYYTSRLHSGVPPSGSGSGSTGDYTLTISLSLPEDFATYYCQQGNLPWTFGQ (including light chain variable region) In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 20E5 as described in WO2014 / 148895A1.

（配列番号５０）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＲＡＳＱＤＩＳＮＹＬＮＷＹＱＱＫＰＧＫＡＶＫＬＬＩＹＹＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＦＣＱＱＧＮＴＬＰＷＴＦＧＱＧＴＫＶＥＩＫＲ（配列番号５２）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 50) and / or the sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLYNWYQQKPGKAVKLLIYYTSRLHSGVPFSSGSGSGDTTYLTISSLQPEDFATYFCQQGNTLWTWTQQGT light chain In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 20E5 as described in WO2014 / 148895A1.

（配列番号５３）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＲＡＳＱＤＩＳＮＹＬＮＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＹＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＧＮＴＬＰＷＴＦＧＱＧＴＫＶＥＩＫＲ（配列番号５１）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 53) and / or the sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLYNWYQQKPGKAPKLLIYYTSRLHSGVPPSGSGSGSTGDYTLTISLSLPEDFATYYCQQGNTLWTWTFGQGTKVEIK In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 20E5 as described in WO2014 / 148895A1.

（配列番号５３）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＲＡＳＱＤＩＳＮＹＬＮＷＹＱＱＫＰＧＫＡＶＫＬＬＩＹＹＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＦＣＱＱＧＮＴＬＰＷＴＦＧＱＧＴＫＶＥＩＫＲ（配列番号５２）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 53) and / or the sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLYNWYQQKPGKAVKLLIYYTSRLHSGVPFSSGSGSGDYTLTIISSLQPEDFATYFCQQGNTLWTWTFGQGT light chain In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 20E5 as described in WO2014 / 148895A1.

（配列番号５４）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＲＡＳＱＤＩＳＮＹＬＮＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＹＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＧＮＴＬＰＷＴＦＧＱＧＴＫＶＥＩＫＲ（配列番号５１）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 54) and / or the sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLYNWYQQKPGKAPKLLIYYTSRLHSGVPFSSGSGSGDTTYLTISSLQPEDFATYYCQQGNLPWTFGQ In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 20E5 as described in WO2014 / 148895A1.

（配列番号５４）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＲＡＳＱＤＩＳＮＹＬＮＷＹＱＱＫＰＧＫＡＶＫＬＬＩＹＹＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＦＣＱＱＧＮＴＬＰＷＴＦＧＱＧＴＫＶＥＩＫＲ（配列番号５２）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン２０Ｅ５の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 54) and / or the sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLYNWYQQKPGKAVKLLIYYTSRLHSGVPFSSGSGSGDYTLTIISSLQPEDFATYFCQQGNTLWTWTQQGT light chain In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 20E5 as described in WO2014 / 148895A1.

ＳＳ（配列番号５５）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＫＡＳＱＤＶＧＡＡＶＡＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＷＡＳＴＲＨＴＧＶＰＳＲＦＳＧＳＧＳＧＴＤＦＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＹＩＮＹＰＬＴＦＧＧＧＴＫＶＥＩＫＲ（配列番号５６）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising SS (SEQ ID NO: 55) and / or the sequence DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRTGVPSRFSGGSGTDFLTTISLQPEDFITYYCQQYINYPLTFGGGTKVGE In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 12H3 described in WO2014 / 148895A1.

（配列番号５５）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＫＡＳＱＤＶＧＡＡＶＡＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＷＡＳＴＲＨＴＧＶＰＤＲＦＳＧＧＧＳＧＴＤＦＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＹＩＮＹＰＬＴＦＧＧＧＴＫＶＥＩＫＲ（配列番号５７）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 55) and / or the sequence DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGGVPRFSGGGSGDFTLTIISSLQPEDFATYCTGQ light chain variable region comprising (SEQ ID NO: 55 In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 12H3 described in WO2014 / 148895A1.

（配列番号５８）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＫＡＳＱＤＶＧＡＡＶＡＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＷＡＳＴＲＨＴＧＶＰＳＲＦＳＧＳＧＳＧＴＤＦＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＹＩＮＹＰＬＴＦＧＧＧＴＫＶＥＩＫＲ（配列番号５６）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 58) and / or the sequence DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGDFTLTIISSLQPEDFATYYCQQYINYPLTFGGGTKVEIK In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 12H3 described in WO2014 / 148895A1.

（配列番号５８）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＫＡＳＱＤＶＧＡＡＶＡＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＷＡＳＴＲＨＴＧＶＰＤＲＦＳＧＧＧＳＧＴＤＦＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＹＩＮＹＰＬＴＦＧＧＧＴＫＶＥＩＫＲ（配列番号５７）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 58) and / or the sequence DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGGVPRFSGGGSGDFTLTIISSLQPEDFATYYCQQYINYPLTFGGGTKVEIK In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 12H3 described in WO2014 / 148895A1.

（配列番号５９）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＫＡＳＱＤＶＧＡＡＶＡＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＷＡＳＴＲＨＴＧＶＰＳＲＦＳＧＳＧＳＧＴＤＦＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＹＩＮＹＰＬＴＦＧＧＧＴＫＶＥＩＫＲ（配列番号５６）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 59) and / or the sequence DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGGSDFLTTISLQPEDFATYYCQQYINYPLTFGGGTKVEIK In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 12H3 described in WO2014 / 148895A1.

（配列番号５９）を含む重鎖可変領域及び／または配列ＤＩＱＭＴＱＳＰＳＳＬＳＡＳＶＧＤＲＶＴＩＴＣＫＡＳＱＤＶＧＡＡＶＡＷＹＱＱＫＰＧＫＡＰＫＬＬＩＹＷＡＳＴＲＨＴＧＶＰＤＲＦＳＧＧＧＳＧＴＤＦＴＬＴＩＳＳＬＱＰＥＤＦＡＴＹＹＣＱＱＹＩＮＹＰＬＴＦＧＧＧＴＫＶＥＩＫＲ（配列番号５７）を含む軽鎖可変領域を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の少なくとも１つ、２つ、３つ、４つ、５つ、または６つの超可変領域（ＨＶＲ）配列を含む。いくつかの実施形態では、本抗体は、ＷＯ２０１４／１４８８９５Ａ１に記載の抗体クローン１２Ｈ３の重鎖可変領域配列及び／または軽鎖可変領域配列を含む。 In some embodiments, the OX40 agonist antibody is an anti-human OX40 agonist antibody as described in WO2014 / 148895A1. In some embodiments, the anti-human OX40 agonist antibody has a sequence

Heavy chain variable region comprising (SEQ ID NO: 59) and / or the sequence DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGGGSGDFLTTIISSLQPEDFATYCTGQ light chain variable region comprising (SEQ ID NO: 59) In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 described in WO2014 / 148895A1. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody clone 12H3 described in WO2014 / 148895A1.

  In some embodiments, the agonist anti-human OX40 antibody is L106 BD (Pharmingen product number 340420). In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody L106 (BD Pharmingen product number 340420). In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody L106 (BD Pharmingen product number 340420).

  In some embodiments, the agonist anti-human OX40 antibody is ACT35 (Santa Cruz Biotechnology, catalog number 20073). In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody ACT35 (Santa Cruz Biotechnology, catalog number 20073). Including. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, catalog number 20073).

  In some embodiments, the OX40 agonist antibody is MEDI6469. In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody MEDI6469.

  In some embodiments, the OX40 agonist antibody is MEDI0562. In some embodiments, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody MEDI0562. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody MEDI0562.

  In some embodiments, the OX40 agonist antibody is an agonist antibody that binds to the same epitope as any of the OX40 agonist antibodies described above.

  In some embodiments, the anti-human OX40 agonist antibody has a functional Fc region. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region is human IgG4. In some embodiments, the anti-human OX40 agonist antibody is engineered to increase effector function (eg, compared to effector function in wild type IgG1). In some embodiments, the antibody has increased binding to Fcγ receptors. In some embodiments, the antibody lacks fucose bound (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% -80%, 1% -65%, 5% -65%, or 20% -40%. In some embodiments, the Fc region comprises a bisected oligosaccharide, eg, a biantennary oligosaccharide attached to an Fc region of the antibody is bisected by GlcNAc. In some embodiments, the antibody has one or more amino acid substitutions that improve ADCC, eg, Fc regions with substitutions at positions 298, 333, and / or 334 (EU numbering of residues). Includes area.

  OX40 agonists useful in the methods described herein are in no way intended to be limited to antibodies. Non-antibody OX40 agonists are contemplated and are well known in the art.

  As described above, OX40L (also known as CD134L) functions as a ligand for OX40. Thus, an agonist that presents some or all of OX40L may function as an OX40 agonist. In some embodiments, the OX40 agonist may comprise one or more extracellular domains of OX40L. An example of the extracellular domain of OX40L can include an OX40 binding domain. In some embodiments, the OX40 agonist may be a soluble form of OX40L that includes one or more extracellular domains of OX40L but lacks other insoluble domains of the protein, such as a transmembrane domain. In some embodiments, the OX40 agonist is a soluble protein comprising one or more extracellular domains of OX40L that can bind to OX40L. In some embodiments, an OX40 agonist can be linked to another protein domain to enhance, for example, its efficacy, half-life, or other desired property. In some embodiments, the OX40 agonist may comprise one or more extracellular domains of OX40L linked to an immunoglobulin Fc domain.

  In some embodiments, the OX40 agonist can be any of the OX40 agonists described in US Pat. No. 7,696,175.

  In some embodiments, the OX40 agonist can be an oligomeric molecule or a multimeric molecule. For example, an OX40 agonist can contain one or more domains that allow the protein to oligomerize (eg, a leucine zipper domain). In some embodiments, the OX40 agonist may comprise one or more extracellular domains of OX40L linked to one or more leucine zipper domains.

  In some embodiments, the OX40 agonist can be any of the OX40 agonists described in European Patent No. EP 0672141 B1.

  In some embodiments, the OX40 agonist can be a trimeric OX40L fusion protein. For example, an OX40 agonist can comprise one or more extracellular domains of OX40L linked to an immunoglobulin Fc domain and a trimerization domain, including but not limited to an isoleucine zipper domain.

  In some embodiments, the OX40 agonist can be any of the OX40 agonists described in International Publication No. WO 2006/121810, such as OX40 immunoadhesin. In some embodiments, the OX40 immunoadhesin can be a trimeric OX40-Fc protein. In some embodiments, the OX40 agonist is MEDI6383.

IV. Antibody Preparation The antibodies described herein are prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the following sections.

  This antibody is directed against a target antigen (that is, PD-L1 (human PD-L1 etc.), OX40 (human OX40 etc.)). Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal suffering from a disorder can provide a therapeutic benefit to the mammal.

In certain embodiments, an antibody provided herein has 1 μM or less, 150 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less. (e.g., ^{10 -8} M or less, ^e.g., 10 -8 ^{M to -13} M, ^e.g., 10 -9 ^{M~10 -13 M)} having a dissociation constant of (Kd).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of the antibody of interest and its antigen, as described by the following assay. The solution binding affinity of the Fab to the antigen is to equilibrate the Fab with a minimal concentration of ( ^125I ) labeled antigen in the presence of a titration series of unlabeled antigen and then capture the bound antigen with an anti-Fab antibody coated plate. (See, eg, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the assay conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg / mL capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate, pH 9.6, It is then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23 ° C.). In a non-adsorbing plate (Nunc number 269620), mix 100 pM or 26 pM [ ¹²⁵ I] antigen with serial dilutions of the desired Fab. The desired Fab is then incubated overnight, but the incubation can be continued for a longer period (eg, about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (eg, 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate is dry, 150 μL / well scintillant (MICROSCINT-20 ™, Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT ™ gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in competitive binding assays.

According to another embodiment, the Kd is BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip of about 10 response units (RU). ) At 25 ° C. using a surface plasmon resonance assay. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was prepared according to the supplier's instructions using N-ethyl-N ′-(3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N -Activate with hydroxysuccinimide (NHS). Dilute the antigen with 10 mM sodium acetate (pH 4.8) to 5 μg / mL (approximately 0.2 μM), then inject at a flow rate of 5 μL / min to provide approximately 10 response units (RU) of coupled protein Get. After the injection of antigen, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, Fab serial dilutions (0.78 nM to 500 nM) were added in PBS with 0.05% polysorbate 20 (TWEEN-20 ™) surfactant (PBST) at 25 ° C. At a flow rate of approximately 25 μL / min. Association rate (k _on ) and dissociation rate (k _off ) by fitting association and dissociation sensorgrams simultaneously using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2) Calculate The equilibrium dissociation constant (Kd) is calculated as the ratio k _off / k _on . For example, Chen et al. , J .; Mol. Biol. 293: 865-881 (1999). If the on-rate exceeds 106M-1 s-1 by the surface plasmon resonance assay described above, the on-rate is measured using a spectrocolorimeter equipped with a stop flow (Aviv Instruments) or 8000 series SLM-AMINCO ™ with a stirred cuvette. Fluorescence emission intensity at 25 ° C. (excitation) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) in the presence of increased concentration of antigen as measured with a spectrophotometer such as ThermoSpectronic = 295 nm, emission = 340 nm, 16 nm bandpass) can be determined by using a fluorescence quench technique that measures the increase or decrease.

(I) Preparation of antigen Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens to generate antibodies. In the case of transmembrane molecules such as receptors, these fragments (eg the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells that express transmembrane molecules can be used as immunogens. Such cells can be derived from natural sources (eg, cancer cell lines) or can be cells that have been transformed by recombinant techniques to express a transmembrane molecule. Other antigens useful in the preparation of antibodies and forms thereof will be apparent to those skilled in the art.

(Ii) Certain Antibody-Based Methods Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Bifunctional or derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl ₂ , or R ¹ N = C = NR, wherein R and R ¹ are different alkyl groups, and the relevant antigen is a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin It may be useful to conjugate to serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

  Animals can be treated with, for example, antigen, Immunize against a protogenic conjugate, or derivative. One month later, the animals are boosted with the initial amount of 1/5 to 1/10 peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. After 7-14 days, animals are bled and the serum is assayed for antibody titer. The animals are boosted until the titer is level. Preferably, the animals are boosted with conjugates of the same antigen but conjugated to different proteins and / or conjugated with different cross-linking reagents. Conjugates can also be made in recombinant cell culture as protein fusions. Aggregating agents such as alum are also preferably used to enhance the immune response.

  The monoclonal antibodies of the present invention are disclosed in Kohler et al. , Nature, 256: 495 (1975) for the first time, see, eg, Hongo et al. , Hybridoma, 14 (3): 253-260 (1995), Harlow et al. , Antibody: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hammerling et al. , Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), and Ni, Xiandai Mianixue, 26 (4): 265-268 (2006) (related to human-human hybridomas). Can be made using the hybridoma method. Additional methods include, for example, the methods described in US Pat. No. 7,189,826 (for production of monoclonal human natural IgM antibodies from hybridoma cell lines). For human hybridoma technology (trioma technology), Volmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods in Findings in Mends in Finland. 91 (2005).

  For various other hybridoma techniques, see, for example, US 2006/258841, US 2006/183877 (fully human antibody), US 2006/059575, US 2005/287149, US 2005/100546, US 2005/026229, and US Pat. No. 7,078,492. And No. 7,153,507. An exemplary protocol for producing monoclonal antibodies using the hybridoma method is described below. In one embodiment, a mouse or other suitable host animal, such as a hamster, produces an antibody that specifically binds to a protein used for immunization or induces lymphocytes that can produce the antibody. To be immunized. The antibody may be administered multiple times with a polypeptide of the invention or fragment thereof and an adjuvant, for example, monophosphoryl lipid A (MPL) / trechrose dicyclinomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.). Produced in animals by subcutaneous (sc) or intraperitoneal (ip) injection. Polypeptides (eg, antigens) or fragments thereof of the present invention can be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. be written. Serum from the immunized animal is assayed for anti-antigen antibodies and optionally booster immunization is performed. Lymphocytes from animals that produce anti-antigen antibodies are isolated. Alternatively, lymphocytes can be immunized in vitro.

  The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells. For example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells that fuse efficiently, support stable high levels of antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium can be used. Exemplary myeloma cells include mouse myeloma lines, such as, for example, Salk Institute Cell Distribution Center, San Diego, Calif. Mouse myeloma lines derived from MOPC-21 and MPC-11 mouse tumors available from the USA, and American Type Culture Collection, Rockville, Md. USA. Mouse myeloma lines derived from SP-2 or X63-Ag8-653 cells available from: Human myeloma and mouse-human heteromyeloma cell lines have also been described for production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984), Brodeur et al., Monoclonal Production Technologies and Applications). 51-63 (Marcel Dekker, Inc., New York, 1987)).

  The hybridoma cells so prepared are seeded and grown in a suitable culture medium, eg, a medium containing one or more substances that inhibit the growth or survival of unfused parent myeloma cells. For example, if the parent myeloma cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically comprises hypoxanthine, aminopterin, and thymidine (HAT medium); These substances block the growth of HGPRT deficient cells. Preferably, see, for example, Even et al. , Trends in Biotechnology, 24 (3), 105-108 (2006), use of serum-free hybridoma cell culture methods to reduce the use of animal-derived sera such as fetal bovine serum Is done.

  Oligopeptides as tools for improving the productivity of hybridoma cell culture are described in Franek, Trends in Monoclonal Antibody Research, 111-122 (2005). Specifically, a standard oligo culture medium enriched with a certain amino acid (alanine, serine, asparagine, proline) or a proteolytic fraction, and a synthetic oligo composed of 3-6 amino acid residues in apoptosis It can be significantly suppressed by peptides. These peptides are present in millimolar or higher concentrations.

  Culture medium in which hybridoma cells are growing can be assayed for production of monoclonal antibodies that bind to the antibodies of the invention. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis. For example, Munson et al. , Anal. Biochem. 107: 220 (1980).

  After hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, clones can be subcloned by limiting dilution procedures and grown by standard methods. See, for example, Goding (see above). Suitable culture media for this purpose include, for example, D-MEM media or RPMI-1640 media. In addition, hybridoma cells can grow in vivo as ascites tumors in animals. Monoclonal antibodies secreted by subclones can be obtained from culture medium, ascites, or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Is preferably separated from One technique for isolating proteins from hybridoma cells is described in US 2005/176122 and US Pat. No. 6,919,436. This method includes using a minimal salt, such as a lyophobic salt, in the binding process, and preferably also using a small amount of organic solvent in the elution process.

(Iii) Library-Derived Antibodies Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having the desired activity (s). For example, various methods are known in the art for generating phage display libraries, such as the method described in Example 3, and screening such libraries for antibodies having the desired binding properties. Further methods are described, for example, in Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, 2001), for example, McCafferty et al. , Nature 348: 552-554, Clackson et al. , Nature 352: 624-628 (1991), Marks et al. , J .; Mol. Biol. 222: 581-597 (1992), Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003), Sidhu et al. , J .; Mol. Biol. 338 (2): 299-310 (2004), Lee et al. , J .; Mol. Biol. 340 (5): 1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004), and Lee et al. , J .; Immunol. Methods 284 (1-2): 119-132 (2004).

  In one particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and recombined randomly in a phage library, which is then described by Winter et al. , Ann. Rev. Immunol. 12: 433-455 (1994), and can be screened for antigen-binding phages. Phages typically display antibody fragments as either single chain Fv (scFv) fragments or Fab fragments. The library from the immunization source provides high affinity antibodies to the immunogen without the need for hybridoma construction. Alternatively, Griffiths et al. , EMBO J, 12: 725-734 (1993), a naïve repertoire has been cloned (eg, from humans) to generate antibodies against a wide range of non-self and self antigens without any immunization. A single source can be provided. Finally, Hoogenboom and Winter, J.A. Mol. Biol. , 227: 381-388 (1992), a naive library clones unrearranged V gene segments from stem cells and uses highly variable CDR3 regions using PCR primers containing random sequences. Can also be made synthetically by achieving rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, No. 2007/0117126, No. 2007/0160598, No. 2007/0237764, No. 2007/0292936, and No. 2009/0002360.

  An antibody or antibody fragment isolated from a human antibody library is considered herein a human antibody or human antibody fragment.

(Iv) Chimeric, humanized, and human antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567 and Morrison et al. , Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region from a mouse, rat, hamster, rabbit, or non-human primate, eg, a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switch” antibody whose class or subclass has changed from the class or subclass of the parent antibody. A chimeric antibody comprises an antigen-binding fragment thereof.

  In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce the immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains in which the HVR, eg, CDR (or portion thereof) is derived from a non-human antibody, and FR (or portion thereof) is derived from a human antibody sequence. . A humanized antibody optionally also includes at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are derived from non-human antibodies (eg, antibodies from which HVR residues are derived), eg, to restore or improve antibody specificity or affinity. Of the corresponding residues.

  Humanized antibodies and methods for producing them are described, for example, in Almagro and Francesson, Front. Biosci. 13: 1619-1633 (2008), for example, see Riechmann et al. , Nature 332: 323-329 (1988), Queen et al. , Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989), US Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al. , Methods 36: 25-34 (2005) (described for SDR (a-CDR) grafts), Padlan, Mol. Immunol. 28: 489-498 (1991) (described in “Resurfacing”), Dall'Acqua et al. , Methods 36: 43-60 (2005) (described for “FR shuffling”), and Osbourn et al. , Methods 36: 61-68 (2005) and Klimka et al. , Br. J. et al. Cancer, 83: 252-260 (2000), which describes a “guided selection” approach to FR shuffling.

  Human framework regions that can be used for humanization include framework regions selected using the “best fit” method (see, eg, Sims et al. J. Immunol. 151: 2296 (1993)). Framework regions derived from consensus sequences of human antibodies of a particular subgroup of light chain variable regions or heavy chain variable regions (eg Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992), And Presta et al., J. Immunol., 151: 2623 (1993)), human mature (somatic mutation) framework regions or human germline framework regions (see, eg, Almagro and Francesson, Front. Biosci. 13:16 19-1633 (2008)), and framework regions obtained from screening of FR libraries (see, eg, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al. , J. Biol. Chem. 271: 22611-22618 (1996)), but is not limited thereto.

  In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

  Human antibodies can be prepared by administering an immunogen to an intact human antibody having a human variable region or a transgenic animal modified to produce an intact antibody in response to an antigen challenge. Such animals typically replace all endogenous immunoglobulin loci or contain all or part of a human immunoglobulin locus that is either extrachromosomal or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 11171-1125 (2005). For example, US Pat. Nos. 6,075,181 and 6,150,584 (described for XENOMOUSE ™ technology), US Pat. No. 5,770,429 (described for HuMab® technology), See also US Pat. No. 7,041,870 (described for KM MOUSE® technology), as well as US Patent Application Publication No. US 2007/0061900 (described for Velocimouse® technology). Intact antibody-derived human variable regions produced by such animals can be further modified, for example, by combining with different human constant regions.

  Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (eg, Kozbor J. Immunol., 133: 3001 (1984), Brodeur et al., Monoclonal Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al. , Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include, for example, US Pat. No. 7,189,826 (described for the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianixue, 26 (4): 265-268 (2006) (human) -The method described in the description of human hybridoma). For human hybridoma technology (trioma technology), Volmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods in Findings in Minds in Finland 91 (2005).

  Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

(V) Antibody fragments Antibody fragments can be produced by traditional means such as enzymatic digestion or by recombinant techniques. Under certain circumstances, there are advantages to using antibody fragments rather than whole antibodies. Smaller sized pieces can allow for rapid clearance and can provide improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.

Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992), and Brennan et al.,). Science, 229: 81 (1985)). However, these fragments can now be produced directly from recombinant host cells. Fab, Fv, and ScFv antibody fragments are all E. coli. expressed in E. coli; Easily mass production of these fragments is possible because they can be secreted from E. coli. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab′-SH fragments can be obtained from E. coli. can be recovered directly from E. coli and chemically coupled to form F (ab ′) ₂ fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ′) ₂ fragments can be isolated directly from recombinant host cell culture. Fab and F (ab ′) ₂ fragments with increased in vivo half-life containing salvage receptor binding epitope residues are described in US Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO93 / 16185, US Pat. Nos. 5,571,894, and 5,587,458. Fv and scFv are the only species that have an intact binding site that lacks a constant region, and therefore they may be suitable for reducing non-specific binding during in vivo use. An scFv fusion protein can be constructed to effect an effector protein fusion at either the amino terminus or the carboxy terminus of the scFv. Antibody Engineering, ed. See Borrebaeck (see above). The antibody fragment can be a “linear antibody” described in, for example, US Pat. No. 5,641,870. Such linear antibodies can be monospecific or bispecific.

(Vi) Multispecific antibodies Multispecific antibodies have binding specificities for at least two different epitopes, which are usually derived from different antigens. While such molecules normally bind only to two different epitopes (ie, bispecific antibodies, BsAbs), antibodies with additional specificity such as trispecific antibodies, as used herein, Included in the expression. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) ₂ bispecific antibodies). In one aspect, a bispecific antibody that binds to OX40 and PD-1 is provided. In one aspect, bispecific antibodies that bind to OX40 and PD-L1 are provided.

  Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, which have different specificities (Millstein et al., Nature, 305 : 537-539 (1983)). Because of the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) can produce a mixture with 10 different antibody molecules, only one of which is the correct duplex. Has a specific structure. Purification of this correct molecule, usually performed by an affinity chromatography step, is somewhat cumbersome and the product yield is low. A similar procedure is described in WO 93/08829 and Traunecker et al. , EMBO J. et al. 10: 3655-3659 (1991).

  One approach for making bispecific antibodies known in the art is the “knobs-in-to-holes” or “prochelance-in-to-cavity” approach. (See, eg, US Pat. No. 5,731,168). In this approach, two immunoglobulin polypeptides (eg, heavy chain polypeptides) each contain an interface. The interface of one immunoglobulin polypeptide interacts with the corresponding interface of the other immunoglobulin polypeptide, thereby allowing the association of two immunoglobulin polypeptides. These interfaces are “knobs” or “prochebalances” (the terms may be used interchangeably herein) located at the interface of one immunoglobulin polypeptide, It can be manipulated to correspond to “holes” or “cavities” located at the interface (these terms may be used interchangeably herein). In some embodiments, the hole is the same or similar size as the knob, and when the two interfaces interact, the knob on one interface can be positioned within the corresponding hole on the other interface Is suitably positioned. Without wishing to be bound by theory, it is believed that this stabilizes the heteromultimer and prefers the formation of heteromultimers over other species, such as homomultimers. In some embodiments, this approach is used to promote heteromultimerization of two different immunoglobulin polypeptides and to include bispecificity comprising two immunoglobulin polypeptides having binding specificities for different epitopes. Antibodies can be made.

^ａアミノ酸の分子量は、水の分子量を差し引いたものである。Ｈａｎｄｂｏｏｋ ｏｆ Ｃｈｅｍｉｓｔｒｙ ａｎｄ Ｐｈｙｓｉｃｓ，４３^ｒｄ ｅｄ．Ｃｌｅｖｅｌａｎｄ，Ｃｈｅｍｉｃａｌ Ｒｕｂｂｅｒ Ｐｕｂｌｉｓｈｉｎｇ Ｃｏ．，１９６１からの値。
^ｂＡ．Ａ．Ｚａｍｙａｔｎｉｎ，Ｐｒｏｇ．Ｂｉｏｐｈｙｓ．Ｍｏｌ．Ｂｉｏｌ．２４：１０７−１２３，１９７２からの値。
^ｃＣ．Ｃｈｏｔｈｉａ，Ｊ．Ｍｏｌ．Ｂｉｏｌ．１０５：１−１４，１９７５からの値。アクセス可能な表面積は、この参考文献の図６〜２０に定義されている。 In some embodiments, knobs can be constructed by replacing small amino acid side chains with larger side chains. In some embodiments, holes can be constructed by replacing large amino acid side chains with smaller side chains. The knob or hole can be present at the original interface or can be introduced synthetically. For example, a knob or hole can be introduced synthetically by modifying the nucleic acid sequence encoding the interface to replace at least one “original” amino acid residue with at least one “import” amino acid residue. Methods for altering the nucleic acid sequence can include standard molecular biology techniques well known in the art. The side chain volumes of various amino acid residues are shown in the table below. In some embodiments, the original residue has a small side chain volume (eg, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine) and the import residue to form the knob is Are naturally occurring amino acids and may include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residue has a large side chain volume (eg, arginine, phenylalanine, tyrosine, and tryptophan) and the import residues to form holes are naturally occurring amino acids. Yes, and can include alanine, serine, threonine, and valine.
Table 1. Characteristics of amino acid residues

The molecular weight of ^a- amino acid is obtained by subtracting the molecular weight of water. Handbook of Chemistry and Physics, 43 ^rd ed. Cleveland, Chemical Rubber Publishing Co. , Values from 1961.
^b A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: Value from 107-123, 1972.
^c C.I. Chothia, J .; Mol. Biol. Values from 105: 1-14, 1975. The accessible surface area is defined in FIGS. 6-20 of this reference.

変異は、元の残基、続いて、Ｋａｂａｔ番号付けシステムを使用した位置、その後、移入残基で表示されている（残基は全て一文字のアミノ酸コードで示されている）。複数の変異は、コロンで区切られている。 In some embodiments, the original residue for forming the knob or hole is identified based on the three-dimensional structure of the heteromultimer. Techniques for obtaining three-dimensional structures known in the art can include x-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these embodiments, CH3 / CH3 interface of human IgG ₁ involves sixteen residues on each domain located on four anti-parallel β strands. Without wishing to be bound by theory, the mutated residues preferably have these two so as to minimize the risk that the knob may be accommodated by the surrounding solvent rather than the compensation hole in the partner CH3 domain. Located on two central antiparallel β-strands. In some embodiments, the mutations that form the corresponding knob and hole in the two immunoglobulin polypeptides correspond to one or more pairs provided in the table below.
Table 2. Example set of corresponding knob and hole-forming mutations

Mutations are indicated by the original residue, followed by the position using the Kabat numbering system, followed by the imported residue (all residues are shown in the single letter amino acid code). Multiple mutations are separated by colons.

  In some embodiments, the immunoglobulin polypeptide comprises a CH3 domain comprising one or more amino acid substitutions listed in Table 2 above. In some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more amino acid substitutions listed in the left column of Table 2, and the right side of Table 2. And a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed in the column.

  After mutating the DNA as described above, a polynucleotide encoding a modified immunoglobulin polypeptide having one or more corresponding knob or hole forming mutations can be prepared using standard recombinant techniques known in the art and It can be expressed and purified using cell lines. For example, U.S. Pat. Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, 7,695,936, 8,216,805, US Publication No. 2013/0089553, and Spiss et al. , Nature Biotechnology 31: 753-758, 2013. Modified immunoglobulin polypeptides are described in E. coli. Prokaryotic host cells such as E. coli or eukaryotic host cells such as CHO cells can be used for production. Immunoglobulin polypeptides with corresponding knobs and holes can be expressed in host cells under co-culture and purified together as heteromultimers, or expressed in a single culture and purified separately, in vitro Can be built with. In some embodiments, two strains of bacterial host cells (one expressing an immunoglobulin polypeptide having a knob and the other expressing an immunoglobulin polypeptide having a hole) are known standards in the art. Are co-cultured using bacterial culture techniques. In some embodiments, the two strains can be mixed in specific ratios, for example, to achieve equal expression levels in culture. In some embodiments, the two strains can be mixed in a ratio of 50:50, 60:40, or 70:30. After the polypeptide is expressed, these cells can be lysed together and the protein extracted. Standard techniques known in the art that allow determination of the abundance of homomultimeric species versus heteromultimeric species may include size exclusion chromatography. In some embodiments, each modified immunoglobulin polypeptide is expressed separately using standard recombinant techniques, and they can be assembled together in vitro. Construction includes, for example, purifying each modified immunoglobulin polypeptide, mixing and incubating them together at equal mass, reducing disulfide (eg, by treatment with dithiothreitol), concentrating, and It can be achieved by reoxidizing the polypeptide. The formed bispecific antibodies can be purified using standard techniques such as cation exchange chromatography and can be measured using standard techniques such as size exclusion chromatography. For a more detailed description of these methods, see Spiss et al. , Nat Biotechnol 31: 753-8, 2013. In some embodiments, modified immunoglobulin polypeptides can be expressed separately in CHO cells and constructed in vitro using the methods described above.

  According to a different approach, antibody variable domains with the desired binding specificity (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. This fusion is preferably a fusion with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. It is typical to have a first heavy chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of these fusions. The immunoglobulin heavy chain fusion, if desired, DNA encoding the immunoglobulin light chain is inserted into a separate expression vector and cotransfected into a suitable host organism. This provides excellent flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments in which the unequal proportion of the three polypeptide chains used during construction provides optimal yield. . However, if the expression of at least two polypeptide chains in equal ratios yields high yields, or the ratios are not particularly important, the sequences encoding two or all three polypeptide chains are It can be inserted into one expression vector.

  In one embodiment of this approach, the bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm ( Providing a second binding specificity). This asymmetric structure provides for the separation of the desired bispecific compound from undesired immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only half of the bispecific molecule provides an easy separation method. It has been found to promote. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al. , Methods in Enzymology, 121: 210 (1986).

In accordance with another approach described in WO 96/27011, the interface between pairs of antibody molecules can be engineered to maximize the proportion of heterodimers recovered from recombinant cell culture. One interface includes at least a portion of the C _H 3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). Compensation “cavities” of the same or similar size as the large side chain (s) are replaced on the interface of the second antibody molecule by replacing the large amino acid side chains with smaller amino acid side chains (eg, alanine or threonine). It is produced. This provides a mechanism for increasing the yield of the heterodimer relative to other undesirable end products such as homodimers.

  Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other can be coupled to biotin. Such antibodies are for the treatment of HIV infection, for example targeting immune system cells to undesired cells (US Pat. No. 4,676,980) (WO91 / 00360, WO92 / 200373, and EP03089). Has been proposed. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are disclosed in US Pat. No. 4,676,980, along with several crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments are also described in this document. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. , Science, 229: 81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to produce F (ab ′) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The produced Fab ′ fragment is then converted to a thionitrobenzoate (TNB) derivative. Thereafter, one of the Fab′-TNB derivatives is reconverted to Fab′-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab′-TNB derivative to form a bispecific antibody. The bispecific antibody produced can be used as an agent for the selective immobilization of enzymes.

Due to recent advances, the E. coli of Fab′-SH fragments that can be chemically coupled to form bispecific antibodies. Direct recovery from E. coli was facilitated. Sharaby et al. , J .; Exp. Med. 175: 217-225 (1992) describe the production of two fully humanized bispecific antibody F (ab ′) ₂ molecules. Each Fab ′ fragment is labeled with E. coli. secreted separately from E. coli and subjected to in vitro directed chemical coupling to form bispecific antibodies.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al. , J .; Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides derived from Fos and Jun proteins were linked to the Fab ′ portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. Hollinger et al. , Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provided an alternative mechanism for making bispecific antibody fragments. These fragments contain a heavy chain variable domain (V _H ) connected to a light chain variable domain (V _L ) by a linker that is too short to allow pairing between two domains on the same chain. Therefore, V _H and V _L domains of one fragment are forced with the complementary V _L domain and V _H domain paired with another fragment, thereby, two antigen binding sites are formed. Another strategy for generating bispecific antibody fragments by using single chain Fv (sFv) dimers has also been reported. Gruber et al, J. MoI. Immunol, 152: 5368 (1994).

  Antibodies with a valency greater than 2 are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. et al. Immunol. 147: 60 (1991).

(Vii) Single Domain Antibodies In some embodiments, the antibodies of the present invention are single domain antibodies. A single domain antibody is a single polypeptide chain that contains all or part of an antibody heavy chain variable domain or all or part of a light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (see Domantis, Inc., Waltham, Mass. See, eg, US Pat. No. 6,248,516 B1). In one embodiment, a single domain antibody consists of all or part of an antibody heavy chain variable domain.

(Viii) Antibody Variants In some embodiments, amino acid sequence modification (s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate changes in the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion from and / or insertion into and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can reach the final construct provided that the final construct has the desired properties. Amino acid modifications may be introduced into the subject antibody when the amino acid sequence of the subject antibody is made.

(Ix) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading “Conservative substitutions”. More substantial changes are shown under the heading “Exemplary substitutions” in Table 1 and are further described below with reference to amino acid side chain classes. Amino acid substitutions are introduced into the antibody of interest and the product can be screened for the desired activity, eg, retention / improvement of antigen binding, reduced immunogenicity, or improvement of ADCC or CDC.
Table 3. Example substitution

Amino acids can be grouped according to general side chain properties.
a. Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile
b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln
c. Acidity: Asp, Glu
d. Basic: His, Lys, Arg
e. Residues affecting chain orientation: Gly, Pro
f. Aromatic: Trp, Tyr, Phe

  Non-conservative substitutions involve exchanging a member of one of these classes for another class.

  One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). In general, the resulting variant (s) selected for further study is a modification of certain biological characteristics (eg, increased affinity, reduced immunogenicity) compared to the parent antibody ( (E.g., improved) and / or have certain biological properties of the parent antibody substantially retained. Exemplary substitutional variants are affinity matured antibodies that can be conveniently generated using, for example, phage display-based affinity maturation techniques such as the techniques described herein. Briefly, one or more HVR residues are mutated and the mutated antibody is displayed on a phage and screened for a specific biological activity (eg, binding affinity).

  Modifications (eg, substitutions) can be made to the HVRs, for example, to improve antibody affinity. Such modifications may result in HVR “hot spots”, ie, residues encoded by codons that undergo frequent mutations during the somatic maturation process (eg, Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)). And / or SDR (a-CDR), and the resulting variant VH or VL is tested for binding affinity. For example, Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, (2001)). It is described in. In some embodiments of affinity maturation, diversity is selected for maturation by any of a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Introduced into the variable gene. A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-oriented approach where several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

  In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs as long as such modifications do not substantially reduce the antibody's ability to bind antigen. For example, conservative modifications that do not substantially reduce binding affinity (eg, conservative substitutions provided herein) can be made with HVRs. Such modifications may be outside of the HVR “hot spot” or SDR. In certain embodiments of the above-described variant VH and VL sequences, each HVR is either unaltered or does not contain more than one, more than two, or more than three amino acid substitutions. It is.

  A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is referred to as “alanine scanning mutagenesis” described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, residues or groups of target residues (eg, charged residues such as arg, asp, his, lys, and glu) are identified and replaced by neutral or negatively charged amino acids (eg, alanine or polyalanine). To determine if the interaction between the antibody and the antigen has been affected. Additional substitutions can be introduced at amino acid positions that are functionally sensitive to the first substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine if they have the desired properties.

  Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions of polypeptide lengths containing between 1 and 100 residues or more, and sequences of single or multiple amino acid residues An example is internal insertion. Examples of terminal insertion include antibodies having an N-terminal methionyl residue. Other insertional variants of the antibody molecule include an N-terminal or C-terminal fusion of the antibody to an enzyme or polypeptide that increases the serum half-life of the antibody (eg, due to ADEPT).

(X) Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

  If the antibody contains an Fc region, the carbohydrate attached to it can be altered. Natural antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide that is attached to Asn297 of the CH2 domain of the Fc region, generally by N-linkage. For example, Wright et al. See TIBTECH 15: 26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and fucose attached to the “trunk” GlcNAc of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be performed to create antibody variants with improved certain properties.

  In one embodiment, an antibody variant comprising an Fc region is provided, wherein the carbohydrate structure attached to the Fc region is depleted or lacks fucose, thereby improving ADCC function. Specifically, antibodies with reduced fucose compared to the amount of fucose of the same antibody produced in wild type CHO cells are contemplated herein. That is, they are less than the amount they would otherwise have if produced by natural CHO cells (eg, CHO cells that produce a natural glycosylation pattern such as CHO cells containing the native FUT8 gene). It has a quantity of fucose. In certain embodiments, the antibody is an antibody in which less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans contain fucose. For example, the amount of fucose in such an antibody can be 1% -80%, 1% -65%, 5% -65%, or 20% -40%. In certain embodiments, the antibody is an antibody that does not contain fucose in any of its N-linked glycans, ie, the antibody has no fucose, no fucose, or afucosylation. Has been. The amount of fucose is measured at Asn297 relative to the sum of all sugar structures bound to Asn297 (eg, complex, hybrid, and high mannose structures) as measured, for example, by MALDI-TOF mass spectrometry as described in WO2008 / 077546. Determined by calculating the average amount of fucose in the sugar chain. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues), but Asn297 is about ± 3 from position 297 due to minor sequence variations within the antibody. It may be located upstream or downstream of the amino acid, ie between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Publication No. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to “defucosylated” or “fucose-deficient” antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazzi et al. J. et al. Mol. Biol. 336: 1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986), US Patent Application No. US 2003/0157108 A1 (Presta, L), and WO 2004/056312 A1 (Adams et al.), Specifically Example 11), and knockout cell lines, such as the alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (eg, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94). 4): 680-688 (2006), and WO2003 / 085,107 see) and the like.

  For example, further provided are antibody variants having a bisected oligosaccharide in which the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are, for example, WO2003 / 011878 (Jean-Mairet et al.), US Pat. No. 6,602,684 (Umana et al.), US2005 / 0123546 (Umana et al.), And Ferrara. et al. , Biotechnology and Bioengineering, 93 (5): 851-861 (2006). Antibody variants having at least one galactose residue within the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.), WO 1998/58964 (Raju, S.), and WO 1999/22764 (Raju, S.).

  In certain embodiments, antibody variants having the Fc region described herein can bind to FcγRIII. In certain embodiments, an antibody variant comprising an Fc region described herein has ADCC activity in the presence of human effector cells, or has a human wild type IgG1 Fc region in the presence of human effector cells. Except that it has increased ADCC activity compared to the same antibody.

(Xi) Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant may comprise a human Fc region sequence (eg, a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (eg, substitution) at one or more amino acid positions.

  In certain embodiments, the present invention has some, but not all, effector functions so that the half-life of the antibody in vivo is important, but certain effector functions (such as complement and ADCC) Contemplate antibody variants that are desirable candidates for applications where) is unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, which are primary cells for mediating ADCC, express Fc (RIII only, whereas monocytes express Fc (RI, Fc (RII, and Fc (RIII. FcR expression in hematopoietic cells) Are summarized in Table 3 of 464 Mitsugu in Ravetch and Kinet, Annu.Rev.Immunol.9: 457-492 (1991) A non-limiting example of an in vitro assay for assessing ADCC activity of a molecule of interest U.S. Pat. No. 5,500,362 (see for example, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)), and Hellstrom, I et. al., Proc. Nat'l Acad. Sci. USA 82: 1499-15. 2 (1985), 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)) or non-radioactive assay methods. (Eg, ACTI ™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA, and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells, or alternatively, the ADCC activity of the molecule of interest is In vivo, example For example, animal models such as those disclosed in Clynes et al.Proc.Nat'l Acad.Sci.USA 95: 652-656 (1998) can be evaluated. Can be confirmed, and therefore lack CDC activity, see, eg, the C1q and C3c binding ELISAs of WO 2006/029879 and WO 2005/100402. CDC assays can be performed (eg, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), Cragg, MS et al., Blood 101: 1045-1052 (2003), And Cragg, M . S. and M.M. J. et al. See Glennie, Blood 103: 2738-2743 (2004)). Methods known in the art can also be used to determine FcRn binding and in vivo clearance / half-life (eg, Petkova, SB et al., Int'l. Immunol. 18 (12) : 1759-1769 (2006)).

  Antibodies with reduced effector function include antibodies having substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737, 056). Such Fc variants include Fc variants having substitutions of two or more of amino acid positions 265, 269, 270, 297, and 327, eg, so-called “DANA” having substitutions of residues 265 and 297 to alanine. ”Fc variants (US Pat. No. 7,332,581).

  Certain antibody variants have been described with improved or reduced binding to FcR (see, eg, US Pat. No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001)).

  In certain embodiments, the antibody variant has one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (residue EU numbering) of the Fc region. Contains the Fc region. In one exemplary embodiment, the antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

  In some embodiments, for example, US Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. et al. Immunol. 164: 4178-4184 (2000), which results in altered (ie, either improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC). To the Fc region.

  Antibodies (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al) with increased half-life involved in the transfer of maternal IgG to the fetus and improved binding to the neonatal Fc receptor (FcRn). , J. Immunol.24: 249 (1994)) is described in US2005 / 0014934A1 (Hinton et al.). Those antibodies include an Fc region having one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413. Including substitutions at one or more of 424, 434, eg, Fc region residue 434 (US Pat. No. 7,371,826). For other examples of Fc region variants, see Duncan & Winter, Nature 322: 738-40 (1988), US Pat. No. 5,648,260, US Pat. No. 5,624,821, and WO 94/29351. Please refer.

(Xii) Antibody Derivatives The antibodies of the present invention can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. In certain embodiments, these moieties suitable for derivatization of the antibody are water soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3, 6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propropylene oxide / ethylene oxide copolymer, Examples include, but are not limited to, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. There. Polyethylene glycol propionaldehyde may be advantageous during manufacture due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization includes the specific properties or functions of the antibody to be improved, whether the derivative of the antibody is used in therapy under defined conditions, etc. Can be determined based on considerations not limited to these.

(Xiii) Vectors, host cells, and recombinant methods Antibodies can be produced using recombinant methods. For recombinant production of the anti-antigen antibody, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or expression. DNA encoding the antibody is easily isolated and using conventional techniques (eg, using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody) And can be sequenced. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(A) Signal sequence component The antibodies of the present invention can be produced recombinantly not only directly but also as a fusion polypeptide with a heterologous polypeptide, which preferably is a mature protein or polypeptide. A signal sequence or other polypeptide having a specific cleavage site at the N-terminus. The heterologous signal sequence selected is preferably one that is recognized and processed (eg, cleaved by a signal peptidase) by the host cell. In a prokaryotic host cell that does not recognize or process the native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, the group of alkaline phosphatase, penicillinase, lpp, or thermostable enterotoxin II leader. In the case of yeast secretion, the natural signal sequence can be, for example, a yeast invertase leader, a factor leader (such as Saccharomyces and Kluyveromyces α-factor leader), or an acid phosphatase leader, C.I. can be replaced by an albicans glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders such as herpes simplex gD signals are available.

(B) Origin of replication Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. In general, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and these include origins of replication or self-replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are available for cloning vectors in mammalian cells. Useful. In general, an origin of replication component is not required for mammalian expression vectors (it can typically be used only because the SV40 origin contains the early promoter).

(C) Selection gene component Expression vectors and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes either (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, (b) store auxotrophic defects, or (c) It encodes a protein that supplies a gene that encodes an essential nutrient that is not available from complex media, such as D-alanine racemase for Bacilli.

  One example of a selection scheme utilizes drugs that stop the growth of host cells. Cells that have been successfully transformed with a heterologous gene confer drug resistance and therefore produce proteins that survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

  Another example of a selectable marker suitable for mammalian cells is DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably antibodies such as primate metallothionein gene, adenosine deaminase, ornithine decarboxylase It enables identification of cellular components for taking up encoding nucleic acids.

  For example, cells transformed with the DHFR gene are identified by culturing the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other co-transformed nucleic acid. A Chinese hamster ovary (CHO) cell line that is deficient in endogenous DHFR activity (eg, ATCC CRL-9096) can be used.

  Alternatively, cells transformed with the GS gene are identified by culturing the transformant in a culture medium containing GS inhibitor L-methioninesulfoximine (Msx). Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acid. A GS selection / amplification system can be used in combination with the DHFR selection / amplification system described above.

  Alternatively, a host cell transformed or co-transformed with a DNA sequence encoding the antibody of interest, a wild type DHFR gene, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH) (specifically Can be selected by cell growth in a medium containing a selective agent for a selectable marker such as an aminoglycoside antibiotic, eg, kanamycin, neomycin, or G418. . See U.S. Pat. No. 4,965,199.

  A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). The trp1 gene provides a selectable marker for a mutant strain of yeast lacking the ability to grow on tryptophan, eg, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of trp1 lesions in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, yeast strains deficient in Leu2 (ATCC 20,622 or 38,626) are complemented by known plasmids with the Leu2 gene.

  In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts. Alternatively, K. as an expression system for large-scale production of recombinant calf chymosin. lactis has been reported. Van den Berg, Bio / Technology, 8: 135 (1990). A stable multicopy expression vector for secretion of mature recombinant human serum albumin by an industrial strain of Kluyveromyces is also disclosed. Freeer et al. Bio / Technology, 9: 968-975 (1991).

(D) Promoter Component Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to the antibody-encoding nucleic acid. Suitable promoters for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter system, alkaline phosphatase promoter, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters for use in bacterial systems also contain a Shine-Dalgarno (SD) sequence that is operably linked to the antibody-encoding DNA.

  Eukaryotic promoter sequences are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N is any nucleotide. The 3 'end of most eukaryotic genes is an AATAAA sequence that can be a signal for the addition of a poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into the nuclear expression vector.

  Examples of promoter sequences suitable for use with yeast hosts include promoters of 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase Phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, glucose phosphate isomerase, and glucokinase.

  Other inducible promoters that are further advantageous inducible promoters for transcription controlled by growth conditions include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde-3- It is the promoter region of phosphate dehydrogenase and enzymes involved in maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

  Antibody transcription from vectors in mammalian host cells includes, for example, polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B From the genome of a virus, such as a virus, simian virus 40 (SV40), or from a heterologous mammalian promoter, such as an actin promoter or an immunoglobulin promoter, can be controlled by a promoter obtained from a heat shock promoter, provided that such promoter is It must be compatible with the system.

  The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in US Pat. No. 4,419,446. Modifications to this system are described in US Pat. No. 4,601,978. For the expression of human β-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus, see Reyes et al. , Nature 297: 598-601 (1982). Alternatively, the rous sarcoma virus long terminal repeat can be used as a promoter.

(E) Enhancer element component Transcription of DNA encoding the antibody of the present invention by higher order eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer (bp 100-270) on the second half of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the second half of the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. Enhancers can be spliced into the vector at positions 5 'or 3' of the antibody coding sequence, but are preferably located 5 'to the promoter.

(F) Transcription termination component Expression vectors used in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) are transcription termination and mRNA stabilization. It also contains sequences necessary for. Such sequences are generally available from the 5 'untranslated region, sometimes the 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments within the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector disclosed therein.

(G) Selection and transformation of host cells Suitable host cells for cloning or expression of DNA in the vectors herein are the prokaryotes, yeasts, or higher order eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria such as Gram negative or Gram positive organisms such as Enterobacteriaceae such as Escherichia such as E. coli. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, eg, Salmonella typhimurium, Serratia, eg, Serratia marcesscans, and Shigella, eg, Bacillus, subtilis and B.M. licheniformis (eg, B. licheniformis 41P disclosed in DD 266, 710 published April 12, 1989), Pseudomonas, eg, P. aeruginosa, and Streptomyces. One preferred E.I. The E. coli cloning host is E. coli. E. coli 294 (ATCC 31, 446). coli B, E.I. E. coli X1776 (ATCC 31,537), and E. coli. Other strains such as E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative rather than limiting.

  Full-length antibodies, antibody fusion proteins, and antibody fragments are specifically cytotoxic drugs (eg, therapeutic antibodies that alone have an effect on tumor cell destruction when glycosylation and Fc effector function are not required) Can be produced in bacteria when conjugated to toxins. Full-length antibodies have a better blood half-life. E. production in E. coli is faster and more cost effective. For expression of antibody fragments and polypeptides in bacteria, see, for example, US Pat. No. 5,648,237 (Carter et. Al.), US Pat. No. 5,789,199 (Jolly et al.), US Pat. See 5,840,523 (Simons et al.), Which describes a translation initiation region (TIR) and signal sequence to optimize expression and secretion. Charleston, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. See also 245-254 (described for expression of antibody fragments in E. coli). After expression, the antibody is isolated from E. coli in the soluble fraction. E. coli cell paste can be isolated, for example, purified by protein A or G column depending on the isotype. Final purification can be performed, for example, similar to the process for purifying antibodies expressed in CHO cells.

  In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Of the lower eukaryotic host microorganisms, Saccharomyces cerevisiae or common baker's yeast are most commonly used. However, Schizosaccharomyces pombe, Kluyveromyces hosts such as K. et al. lactis, K. et al. fragilis (ATCC 12,424), K.M. bulgaricus (ATCC 16,045), K. et al. wickeramii (ATCC 24,178), K.K. waltii (ATCC 56,500), K.M. Drosophilarum (ATCC 36,906), K.M. thermotolerans, and K.K. marxianus et al., yarrowia (EP402, 226), Pichia pastoris (EP183,070), Candida, Trichoderma reesia (EP244, 234), Neurospora crassa, Swannimyces, e.g., occlusion , And Aspergillus hosts such as A. nidulans and A.N. Several other genera, species, and strains such as niger are also generally available and useful herein. For a review discussing the use of yeast and filamentous fungi for the production of therapeutic proteins, see, eg, Gerngross, Nat. Biotech. 22: 1409-1414 (2004).

  Certain fungal and yeast strains can be selected in which the glycosylation pathway is “humanized”, resulting in the production of antibodies having a partial or fully human glycosylation pattern. For example, Li et al. Nat. Biotech. 24: 210-215 (2006) (described for humanization of the glycosylation pathway in Pichia pastoris), and Gerngross et al. (See above).

  Suitable host cells for the expression of glycosylated antibody can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, as well as hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (corresponding to insect cells) and x Have been identified. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, such viruses specifically include Spodoptera frugiperda. For transfection of cells, it can be used as a virus herein according to the invention.

  Cotton, corn, potato, soybean, petunia, tomato, Leninaceae, M. truncula, and tobacco plant cell cultures can also be utilized as hosts. For example, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (transformers) See PLANTIBODIES ™ technology for the production of antibodies in transgenic plants).

Vertebrate cells can be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 strain (COS-7, ATCC CRL 1651) transformed with SV40, human embryonic kidney strain (293 subcloned for growth in suspension culture or 293 cells, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4, Mother, Biol. Reprod. 23: 243-251). (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC) CCL 34), buffer roll Liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human lung cells (Hep G2, HB 8065), mouse mammary tumors (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Anals NY Acad. Sci. 383: 44-68 (1982)), MRC 5 cells, FS4 cells, and human liver cancer (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, such as DHFR ^- CHO cells (Urrub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)), and bone marrow And tumor cell lines such as NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. See 255-268.

  A host cell is transformed with an expression vector or cloning vector as described above for antibody production and modified as appropriate for induction of a promoter, selection of a transformation pair, or amplification of a gene encoding the desired sequence. In a nutrient medium.

(H) Culture of host cells The host cells used to produce the antibodies of the invention are cultured in a variety of media. Commercially available media such as Ham F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for host cell culture. In addition, Ham et al., Meth. Enz.58: 44 (1979), Barnes et al., Anal.Biochem.102: 255 (1980), US Patent No. 4,767,704, ibid. No. 4,657,866, No. 4,927,762, No. 4,560,655, or No. 5,122,469, WO90 / 03430, WO87 / 00195, or US Patent Reissue No. 30 , 985 may be used as the culture medium for the host cells. Any of these may be hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES) as necessary. ), Nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ™ drugs), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art Culture conditions, such as temperature, pH, etc., are those already used in the host cell selected for expression. And will be apparent to those skilled in the art.

(Xiv) Purification of antibodies When using recombinant techniques, the antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris (either host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al. Bio / Technology 10: 163-167 (1992). Describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors such as PMSF to inhibit proteolysis may be included in any of the foregoing steps, and antibiotics may be included to block the growth of exogenous contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being typical Is one of the preferred purification steps. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand binds is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a C _H 3 domain, Bakerbond ABX ™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, eg fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin, anion or cation exchange resin (polyaspartic acid column Etc.) SEPHAROSE ™ chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody recovered.

  In general, various methodologies for preparing antibodies for use in research, testing, and clinical use are established in the art, consistent with the above-described methodologies, and / or aimed by one of ordinary skill in the art. It is considered appropriate for a particular antibody.

C. Selection of biologically active antibodies Antibodies produced as described above may be subjected to one or more “biological activity” assays to select antibodies having beneficial properties from a therapeutic perspective, Alternatively, a formulation and conditions that retain the biological activity of the antibody can be selected. The antibody can be tested for its ability to bind to the antigen from which it is produced. For example, methods known in the art (eg, ELISA, Western blot, etc.) can be used.

  For example, in the case of an anti-PDL1 antibody, the antigen binding properties of the antibody can be assessed in an assay that detects the ability to bind to PDL1. In some embodiments, binding of the antibody can be determined, for example, by saturation binding, ELISA, and / or competition assays (eg, RIA). The antibodies can also be subjected to other biological activity assays, for example, to assess their effectiveness as therapeutic agents. Such assays are known in the art and depend on the target antigen and intended use of the antibody. For example, the biological effects of PD-L1 blockade by the present antibodies are CD8 + T cells, lymphocytic choriomeningitis virus (LCMV) mouse models, and / or syngeneic tumor models such as US Pat. No. 8,217,149. In the model described in the issue.

  To screen for antibodies that bind to specific epitopes on the antigen of interest (eg, antibodies that block the binding of an exemplary anti-PDL1 antibody to PD-L1), routine cross-blocking assays such as Antibodies, The assay described in A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) may be performed. Alternatively, epitope mapping, eg, Champe et al. , J .; Biol. Chem. 270: 1388-1394 (1995) can be used to determine if the antibody binds to the epitope of interest.

  In one aspect, an assay for identifying the anti-OX40 antibody having biological activity is provided. Biological activities include, for example, OX40 binding (eg, human and / or cynomolgus OX40 binding), increased OX40-mediated signaling (eg, increased NFkB-mediated transcription), cells expressing human OX40 (eg, T cell) depletion, effector T cell function enhancement (eg, CD4 + effector T cells, CD8 + effector T cells) (eg, increased effector T cell proliferation and / or increased cytokine production by effector T cells (eg, gamma interferon) )) Enhancement of memory T cell function (eg, CD4 + memory T cells) (eg, due to increased memory T cell proliferation and / or increased cytokine production by memory T cells (eg, gamma interferon)), regulatory T Inhibition of cell function ( Eg to effector T cell function (e.g., CD4 + effector T cell function, CD8 + effector T cell function) due to the reduction of Treg suppression) can be mentioned. Antibodies having such biological activity in vivo and / or in vitro are provided.

  In certain embodiments, the antibodies of the invention are tested for such biological activity.

  Methods known in the art can be used to assay T cell costimulation and exemplary methods are disclosed herein. For example, T cells (eg, memory or effector T cells) can be obtained from peripheral leukocytes (eg, isolated from human whole blood using Ficoll gradient centrifugation). Memory T cells (eg, CD4 + memory T cells) or effector T cells (eg, CD4 + Teff cells) can be isolated from PBMC using methods known in the art. For example, a Miltenyi CD4 + memory T cell isolation kit or a Miltenyi naive CD4 + T cell isolation kit can be used. Isolated T cells are cultured in the presence of antigen presenting cells (eg, irradiated L cells expressing CD32 and CD80) and activated by the addition of anti-CD3 antibody in the presence or absence of OX40 agonist antibody. The The effect of agonist OX40 antibody on T cell proliferation can be measured using methods well known in the art. For example, the CellTiter Glo kit (Promega) may be used and the results are read with a multi-label reader (Perkin Elmer). The effect of agonist OX40 antibody on T cell function can also be determined by analysis of cytokines produced by T cells. In one embodiment, production of interferon gamma by CD4 + T cells is determined, for example, by measuring interferon gamma in the cell culture supernatant. Methods for measuring interferon gamma are well known in the art.

  Treg cell function can be assayed using methods known in the art, and exemplary methods are disclosed herein. In one example, the ability of Tregs to inhibit effector T cell proliferation is assayed. T cells are isolated from human whole blood using methods known in the art (eg, by isolating memory T cells or naïve T cells). Purified CD4 + naive T cells are labeled (eg, with CFSE) and purified Treg cells are labeled with different reagents. Irradiated antigen presenting cells (eg, L cells expressing CD32 and CD80) are co-cultured with labeled and purified naive CD4 + T cells and purified Tregs. Co-cultures are activated using anti-CD3 antibody and tested in the presence or absence of agonist OX40 antibody. After a suitable time (eg, 6 days co-culture), the level of CD4 + naive T cell proliferation is followed by dye dilution at reduced labeling staining (eg, reduced CFSE labeling staining) using FACS analysis. Is done.

  OX40 signaling can be assayed using methods well known in the art, and exemplary methods are disclosed herein. In one embodiment, a transgenic cell is generated that expresses a reporter gene comprising an NFkB promoter fused to human OX40 and a reporter gene (eg, beta luciferase). Addition of OX40 agonist antibody to those cells results in an increase in NFkB transcription, which is detected using an assay for a reporter gene.

  Phagocytosis can be assayed using, for example, monocyte-derived macrophages, or U937 cells (a human histiocytic lymphoma cell line with mature macrophage morphology and characteristics). OX40 expressing cells are added to monocyte-derived macrophages or U937 cells in the presence or absence of anti-OX40 agonist antibodies. After culturing these cells for a suitable period, the rate of phagocytosis was tested for the proportion of cells that double-stained the markers of 1) macrophages or U937 cells and 2) OX40-expressing cells, and this was determined for OX40-expressing cells. Determined by dividing by the total number of cells exhibiting the marker (eg, GFP). Analysis can be done by flow cytometry. In another embodiment, the analysis can be performed by fluorescence microscopy analysis.

  ADCC can be assayed using, for example, methods well known in the art. Exemplary methods are described in the definitions section and exemplary assays are disclosed in the examples. In some embodiments, the level of OX40 is characterized on OX40 expressing cells used for testing in an ADCC assay. Cells can be stained with a detectably labeled anti-OX40 antibody (eg, PE label), after which the fluorescence level can be determined using flow cytometry and the results can be presented as the mean median fluorescence intensity (MFI) . In another embodiment, ADCC can be analyzed by CellTiter Glo assay kit and cell viability / cytotoxicity can be determined by chemiluminescence.

The binding affinity of various antibodies to the two allotypes (F158 and V158) of FcγRIA, FcγRIIA, FcγRIIB, and FcγRIIIA can be measured in an ELISA-based ligand binding assay using the respective recombinant Fcγ receptors. Purified human Fcγ receptor is expressed as a fusion protein containing the extracellular domain of the receptor γ chain linked to a C-terminal Gly / 6xHis / glutathione S-transferase (GST) polypeptide tag. The binding affinity of antibodies to their human Fcγ receptor is assayed as follows. For the low affinity receptors, FcγRIIA (CD32A), FcγRIIB (CD32B), and FcγRIIIA (CD16), the two allotypes F-158 and V-158, the antibody is an antibody: cross-linked F (ab ′) ₂ It can be tested as a multimer by cross-linking with the F (ab ′) 2 fragment of goat anti-human kappa chain (ICN Biomedical, Irvine, Calif.) At an appropriate molar ratio of 1: 3. Plates are coated with anti-GST antibody (Genentech) and blocked with bovine serum albumin (BSA). After washing with phosphate buffered saline (PBS) containing 0.05% Tween-20 using an ELx405 ™ plate washer (Biotek Instruments, Winooski, VT), Fcγ receptor was 25 ng / well. Add to plate and incubate for 1 hour at room temperature. After the plates were washed, serial dilutions of test antibody were added as multimeric complexes and the plates were incubated for 2 hours at room temperature. After the plate is washed to remove unbound antibody, the antibody bound to the Fcγ receptor is a horseradish peroxidase (HRP) from goat anti-human F (ab ′) ₂ (Jackson ImmunoResearch Laboratories, West Grove, PA). The conjugate F (ab ′) ₂ fragment is detected, followed by the addition of the substrate tetramethylbenzidine (TMB) (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Depending on the Fcγ receptor being tested, the plate is incubated at room temperature for 5-20 minutes to allow color development. The reaction was terminated with 1M H ₃ PO ₄ and the absorbance at 450 nm was measured with a microplate reader (SpectraMax® 190, Molecular Devices, Sunnyvale, Calif.). A dose response binding curve is generated by plotting the mean absorbance value from duplicate antibody dilutions against the concentration of antibody. The effective concentration of the antibody at which 50% of the maximum response from binding to the Fcγ receptor (EC ₅₀ ) was detected was fitted to a binding curve in a 4-parameter equation using SoftMax Pro (Molecular Devices). It was decided later.

  Cells for use in any of the above in vitro assays include cells or cell lines that naturally express OX40 or have been engineered to express OX40. Such cells include activated T cells, Treg cells, and activated memory T cells that naturally express OX40. Such cells also include cell lines that do not normally express OX40 but express OX40 and cell lines transfected with a nucleic acid encoding OX40. Exemplary cell lines provided herein for use in any of the in vitro assays described above include transgenic BT474 cells (human breast cancer cell line) that express human OX40.

  It will be appreciated that the immunoconjugates of the invention can be used in place of or in addition to anti-OX40 antibodies to perform any of the assays described above.

  It is understood that an anti-OX40 antibody and an additional therapeutic agent (eg, a PD-1 axis binding agent (eg, anti-PD-1 or anti-PD-L1 antibody) can be used to perform any of the above assays. Is done.

D. Pharmaceutical Composition and Formulation A pharmaceutical comprising a PD-1 axis binding antagonist and / or an antibody described herein (such as an anti-PD-L1 antibody or an anti-human OX40 agonist antibody, and a pharmaceutically acceptable carrier). Compositions and formulations are also provided herein.

  The pharmaceutical compositions and formulations described herein comprise an active ingredient (such as an antibody or polypeptide) having a desired purity in one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition). , Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, including buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants Preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; Resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptide; protein, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone ; Mino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose , Mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (eg, Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG), It is not limited to. Exemplary pharmaceutically acceptable carriers herein include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP) such as human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20. (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, such as rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

  An exemplary lyophilized antibody formulation is described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006/044908, the latter formulation including a histidine-acetate buffer.

  The compositions and formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are preferably present in combination in amounts that are effective for the purpose intended.

  The active ingredients are contained in colloid drug delivery systems (e.g. Liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. et al. Ed. (1980).

  Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semi-permeable matrices of solid hydrophobic polymers containing the present antibodies, which are in the form of molded articles such as films or microcapsules. The formulations used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration through sterile filtration membranes.

IV. Methods of Treatment A method of treating cancer or delaying its progression in an individual is disclosed herein, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody). Provided. In some embodiments, the treatment results in a sustained response in the individual after cessation of treatment. The methods described herein may find use in the treatment of conditions where enhanced immunogenicity is desired, such as increased tumor immunogenicity for the treatment of cancer. Also provided herein is a method of enhancing immune function in an individual having cancer, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody). In a further aspect, provided herein are methods for treating an infection (eg, a bacterial or viral or other pathogen infection). In some embodiments, the infection is a viral infection and / or a bacterial infection. In some embodiments, the infection is a pathogen infection. In some embodiments, the infection is an acute infection. In some embodiments, the infection is a chronic infection.

  Any of the PD-1 axis binding antagonists and OX40 binding agonists known in the art or described herein can be used in these methods.

  In some embodiments, the individual is a human.

  In some embodiments, the individual has been treated with an OX40 binding agonist therapy prior to combination treatment with a PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody).

  In some embodiments, the individual has a cancer that is resistant (proven to be resistant) to one or more PD-1 axis antagonists. In some embodiments, resistance to a PD-1 axis antagonist comprises cancer recurrence or refractory cancer. Recurrence can refer to the reappearance of cancer at the site of onset or new site after treatment. In some embodiments, resistance to a PD-1 axis antagonist comprises progression of cancer during treatment with the PD-1 axis antagonist. In some embodiments, resistance to a PD-1 axis antagonist comprises a cancer that does not respond to treatment. The cancer can be resistant at the start of treatment or can become resistant during treatment. In some embodiments, the cancer is early cancer or late cancer.

  In another aspect, the individual has a cancer that expresses a PD-L1 biomarker (eg, shown to be expressed in a diagnostic test). In some embodiments, the patient's cancer expresses a low PD-L1 biomarker. In some embodiments, the patient's cancer expresses a high PD-L1 biomarker. In some embodiments of any of these methods, assays, and / or kits, the PD-L1 biomarker is not present in the sample when included in 0% of the sample.

  In some embodiments of any of these methods, assays, and / or kits, the PD-L1 biomarker is present in the sample when it is present in greater than 0% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 1% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 5% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 10% of the sample.

  In some embodiments of any of these methods, assays, and / or kits, the PD-L1 biomarker is FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, Dot blot method, immunodetection method, HPLC, surface plasmon resonance, optical spectroscopy, mass spectroscopy, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, and FISH , As well as combinations thereof, are detected in the sample using a method selected from the group consisting of.

  In some embodiments of any of these methods, assays, and / or kits, the PD-L1 biomarker is detected in the sample by protein expression. In some embodiments, protein expression is determined by immunohistochemistry (IHC). In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody. In some embodiments, the PD-L1 biomarker is detected as a weak staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected as moderate staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected as a strong staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected in tumor cells, tumor infiltrating immune cells, stromal cells, and any combination thereof. In some embodiments, the staining is membrane staining, cytoplasmic staining, or a combination thereof.

  In some embodiments of any of these methods, assays, and / or kits, the absence of PD-L1 biomarker is detected as absence of staining or no staining in the sample. In some embodiments of any of these methods, assays, and / or kits, the presence of the PD-L1 biomarker is detected as any staining in the sample.

  In some embodiments, the combination therapies of the invention comprise the administration of a PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody). The PD-1 axis binding antagonist and OX40 binding agonist can be administered in any suitable manner known in the art. For example, the PD-1 axis binding antagonist and the OX40 binding agonist can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the PD-1 axis binding antagonist is present in a separate composition from the OX40 binding agonist. In some embodiments, the PD-1 axis binding antagonist is present in the same composition as the OX40 binding agonist.

  The PD-1 axis binding antagonist and OX40 binding agonist (eg, an anti-human OX40 agonist antibody) can be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axis binding antagonist is intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, by implantation, by inhalation, intrathecal, intraventricular, or It is administered intranasally. In some embodiments, the OX40 binding agonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecal, intraventricular, or intranasal. Is done. An effective amount of a PD-1 axis binding antagonist and OX40 binding agonist can be administered for the prevention or treatment of a disease. Suitable dosages of a PD-1 axis binding antagonist and / or OX40 binding agonist (eg, an anti-human OX40 agonist antibody) are determined by the type of disease being treated, the type of PD-1 axis binding antagonist and OX40 binding agonist, the type of disease. It can be determined based on the severity and course, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of the attending physician. In some embodiments, the combination treatment of an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) and a PD-1 axis binding antagonist (eg, an anti-PD-1 or anti-PDL1 antibody) is synergistic, thereby The effective dose of the OX40 binding agent (eg, anti-human OX40 agonist antibody) in combination is reduced compared to the effective dose of the OX40 binding agent (eg, anti-human OX40 agonist antibody) as a single agent.

  As a general proposition, a therapeutically effective amount of antibody administered to a human will range from about 0.01 to about 50 mg / kg patient body weight, regardless of one or more administrations. In some embodiments, the antibody used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg, for example, administered daily. In some embodiments, the antibody is administered at 15 mg / kg. However, other dosage regimens may be useful. In one embodiment, the anti-PDL1 antibody described herein is about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about Administered to humans at doses of 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg. This dose can be administered as a single dose, such as an infusion, or as multiple doses (eg, 2 or 3 doses). The dose of the antibody administered in combination therapy can be reduced compared to single agent therapy. The progress of this therapy is easily monitored by conventional techniques.

  In some embodiments, these methods may further comprise additional therapies. Further therapies are radiation therapy, surgery (eg, tumorectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or as described above It can be a combination of The additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or antimetastatic agent. In some embodiments, the additional therapy is the administration of a side effect limiting agent (eg, an agent intended to reduce the occurrence and / or severity of treatment side effects, such as an antiemetic agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy that targets the PI3K / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. In some embodiments, the additional therapy is CTLA-4 (also known as CD152), such as blocking antibodies, ipilimumab (also known as MDX-010, MDX-101, or Yervoy®), tremelimumab (also known as ticilimumab) Or CP-675,206), antagonists directed against B7-H3 (also known as CD276), such as blocking antibodies, MGA271, antagonists directed against TGFbeta, such as meterimumab (also known as CAT-192) ), Fresolimumab (also known as GC1008), or LY2157299, a treatment involving adoptive transfer of T cells (eg, cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), a dominant negative TGF beta receptor, such as Dominant negative T Treatments that include adoptive transfer of T cells containing Fbeta type II receptors, treatments that include the HERCREEM protocol (see, for example, ClinicalTrials.gov Identifier NCT00889954), agonists directed against CD137 (also known as TNFRSF9, 4 -1BB, or ILA), eg, an activating antibody, urelumab (also known as BMS-663513), an agonist directed against CD40, eg, an activating antibody, CP-870893, a different anti-OX40 antibody (eg, AgonOX) Agonists (also known as CD134) directed against OX40 administered in combination with, for example, activated antibodies, agonists directed against CD27, such as activated antibodies, CDX-1127, indoleamine-2 3-dioxygenase (IDO), 1-methyl-D-tryptophan (also known as 1-D-MT), antibody-drug conjugate (in some embodiments, including metansin or monomethyl auristatin E (MMAE)) , Anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599), trastuzumab emtansine (also known as T-DM1, adtrastuzumab emtansine, or KADCYLA®, Genentech), DMUC5754A, endothelin B receptor (EDNBR) Antibody-drug conjugates targeted to, for example, antibodies directed against EDNBR conjugated to MMAE, angiogenesis inhibitors, antibodies directed against VEGF, such as VEGF-A, Bebashi Mabs (also known as AVASTIN®, Genentech), antibodies directed against Angiopoietin 2 (also known as Ang2), MEDI3617, antineoplastic drugs, drugs targeting CSF-1R (also known as M-CSFR or CD115), anti-CSF-1R (also known as IMC-CS4), interferons such as interferon alpha or interferon gamma, roferon-A, GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, Sargramostim, or Leukine®, IL-2 (also known as Aldesleukin or Proleukin®), IL-12, an antibody that targets CD20 (in some embodiments, this targets CD20) The antibody Vinutuzumab (also known as GA101 or Gaziva®) or rituximab, an antibody that targets GITR (in some embodiments, this antibody that targets GITR is TRX518), a cancer vaccine (some In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine, and in some embodiments, the peptide cancer vaccine is a multivalent peptide, Multipeptide, peptide cocktail, hybrid peptide, or peptide pulsed dendritic cell vaccines (see, eg, Yamada et al. , Cancer Sci, 104: 14-21, 2013)), in combination with adjuvants, TLR agonists such as poly-ICLC (also known as Hitonol®), LPS, MPL, or CpG ODN, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonist, IL-4 antagonist, IL-13 antagonist, HVEM antagonist, ICOS agonist, eg, by administration of ICOS-L or against ICOS -Directed agonist antibody, treatment targeting CX3CL1, treatment targeting CXCL10, treatment targeting CCL5, LFA-1 or ICAM1 agonist, selectin agonist, targeted therapy, B-Raf inhibitor, vemurafenib (also known as , Zelboraf®, dabrafenib (also known as Tafinlar®), erlotinib (also known as Tarceva®), MEK inhibitors such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2), kobimetinib (Aka GDC-0973 or XL-518), trametinib (aka Mekinist®), K-Ras inhibitor, c-Met inhibitor, onartuzumab (aka MetMAb), Alk inhibitor, AF802 (aka, CH5424242 or alectinib), phosphatidylinositol 3-kinase inhibitor (PI3K), BKM120, idealarib (aka GS-1101 or CAL-101), perifosine (aka KRX-0401), Akt, MK22 6, GSK690693, GDC-0941, mTOR inhibitor, sirolimus (also known as rapamycin), temsirolimus (also known as CCI-779 or Torisel (registered trademark)), everolimus (also known as RAD001), reidaforimus (also known as AP-23573) , MK-8669, or Deforolimus), OSI-027, AZD8055, INK128, dual PI3K / mTOR inhibitor, XL765, GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-0469150, P 05212384 (also known as PKI-587) The additional therapy may be one or more of the chemotherapeutic agents described herein.

  The efficacy of any of the methods described herein (e.g., combination therapy including co-administration of an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist) is a clinical model or a preclinical model, etc. Can be tested in various models known in the art. Suitable preclinical models are exemplified herein, and further include ID8 ovarian cancer, GEM model, B16 melanoma, RENCA renal cell carcinoma, CT26 colorectal cancer, MC38 colorectal cancer, and Cloudman melanoma cancer model Can be mentioned, but is not limited thereto.

The efficacy of any of the methods described herein (eg, combination therapy including co-administration of an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist) is non-small cell lung cancer, pancreatic ductal gland It can be tested in GEM models that develop tumors, including but not limited to GEM models of cancer, or melanoma. For example, mice expressing Kras ^G12D in a p53 ^null background after adenovirus recombinase treatment (Jackson, ^EL , et al. (2001) Genes Dev. 15 (24): 3243-8 ( ^Explanation of Kras ^G12D ) and Lee, CL, et al. (2012) Dis.Model Mech.5 (3): 397-402 (FRT-mediated p53 ^null allele) is a preclinical model for non-small cell lung cancer. Can be used. As another example, mice expressing Kras ^G12D in a p16 / p19 ^null background (Jackson, ^EL , et al. (2001) Genes Dev. 15 (24): 3243-8 (Description of Kras ^G12D ) and Aguirre, AJ, et al. (2003) Genes Dev. 17 (24): 3112-26 (p16 / p19 ^null allele) is a preclinical model of pancreatic ductal adenocarcinoma (PDAC). Can be used. As a further example, mice with melanocytes expressing Braf ^V600E in a melanocyte-specific PTEN ^null background after inducible (eg 4-OHT treatment) recombinase treatment (Dankort, D., et al. (2007) Genes Dev 21 (4): 379-84 (Description of Braf ^V600E ) and Trotman, LC, et al. (2003) PLoS Biol.1 (3): E59 (PTEN ^null allele)). Can be used as a preclinical model of melanoma. For any of these exemplary models, after developing a tumor, the mice are treated in a treatment group receiving a combination treatment with an anti-PDL1 and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) or a treatment receiving a control treatment. Randomly mobilized to the group. Tumor size (eg, tumor volume) is measured during the course of treatment and overall survival is also monitored.

  In another aspect, provided herein is a method of enhancing immune function in an individual having cancer, comprising co-administering an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist.

  In some embodiments of the disclosed methods, the cancer (in some embodiments, a sample of a patient's cancer being tested using a diagnostic test) has an elevated level of T cell infiltration. As used herein, cancer T cell infiltration may refer to the presence of T cells, such as tumor infiltrating lymphocytes (TILs) in cancer tissue or otherwise associated with cancer tissue. It is known in the art that T cell infiltration can be associated with improved clinical outcome in certain cancers (eg, Zhang et al., N. Engl. J. Med. 348 (3): 203-213). (See 2003).

  However, T cell depletion is also a major immunological feature of cancer, and many tumor infiltrating lymphocytes (TIL) express high levels of inhibitory co-receptors and lack the ability to produce effector cytokines (Wherry). , E. J. Nature immunology 12: 492-499 (2011), Rabinovich, GA, et al., Annual review of immunology 25: 267-296 (2007)). In some embodiments of the disclosed methods, the individual has a T cell dysfunction disorder. In some embodiments of the disclosed methods, the T cell dysfunction disorder is characterized by a decrease in T cell anergy, or the ability to secrete, proliferate, or perform cytolytic activity. In some embodiments of the disclosed methods, the T cell dysfunction disorder is characterized by T cell depletion. In some embodiments of the disclosed methods, the T cells are CD4 + and CD8 + T cells. Without being bound by theory, OX40 binding agonist treatment increases priming, activation, and / or proliferation of T cells (eg, CD4 + T cells, CD8 + T cells, memory T cells) compared to prior to administration of the combination. Can be. In some embodiments, the T cells are CD4 + and / or CD8 + T cells.

  In some embodiments of the disclosed methods, the cancer (in some embodiments, a patient cancer sample is tested using a diagnostic test) has a low level of T cell infiltration. In some embodiments, the cancer (in some embodiments, a patient cancer sample is tested using a diagnostic test) does not have a detectable T cell infiltrate. In some embodiments, the cancer is a non-immunogenic cancer (eg, non-immunogenic colorectal cancer and / or ovarian cancer). Without being bound by theory, OX40 binding agonist treatment increases priming, activation, and / or proliferation of T cells (eg, CD4 + T cells, CD8 + T cells, memory T cells) compared to prior to administration of the combination. Can be.

In some embodiments of the disclosed methods, activated CD4 and / or CD8 T cells in the individual produce γ-IFN ⁺ and / or produce CD4 and / or CD8 T cells compared to prior to administration of the combination. Alternatively, it is characterized by enhanced cytolytic activity. γ-IFN ⁺ can be measured by any means known in the art including, for example, cell fixation, permeabilization, and intracellular cytokine staining (ICS) with staining with antibodies to γ-IFN. Cytolytic activity can be measured by any means known in the art, for example using a cell killing assay in which effectors and target cells are mixed.

  In some embodiments, the CD8 + T cell has, for example, the presence of CD8b expression (eg, using rtPCR, eg, Fluidigm) (also known as CD8b, T cell surface glycoprotein CD8 beta chain, CD8 antigen, alpha polypeptide). p37, accession number NM_172213). In some embodiments, the CD8 + T cells are derived from peripheral blood. In some embodiments, the CD8 + T cells are derived from a tumor.

  In some embodiments, the Treg cells, for example, have the presence of Fox3p expression (eg, using rtPCR, eg, Fluidigm) (Foxp3 alias, forkhead box protein P3, scurfin, FOXP3delta7 , Immune deficiency, multi-glandular endocrine disorder, enteropathy, X-linked, accession number NM — 014009). In some embodiments, the Treg is from peripheral blood. In some embodiments, the Treg cells are derived from a tumor.

  In some embodiments, inflammatory T cells are characterized, for example, by the presence of Tbet and / or CXCR3 expression (eg, using rtPCR, eg, Fluidigm). In some embodiments, the inflammatory T cells are derived from peripheral blood. In some embodiments, the inflammatory T cells are derived from a tumor.

  In some embodiments of the disclosed methods, the CD4 and / or CD8 T cells exhibit an increase in the release of a cytokine selected from the group consisting of IFN-γ, TNF-α, and interleukin. Cytokine release can be achieved by any means known in the art, eg, Western blot, ELISA, or immunization to detect the presence of released cytokines in a sample containing CD4 and / or CD8 T cells. It can be measured using a histochemical assay.

In some embodiments of the disclosed methods, the CD4 and / or CD8 T cells are effector memory T cells. In some embodiments of the disclosed methods, the CD4 and / or CD8 effector memory T cells are characterized by having a CD44 ^high CD62L ^low expression. Expression of CD44 ^high CD62L ^low can be achieved by any means known in the art, eg, preparing a single cell suspension of tissue (eg, cancer tissue) and using commercially available antibodies against CD44 and CD62L. And can be detected by surface staining and flow cytometry. In some embodiments of the disclosed methods, the CD4 and / or CD8 effector memory T cells express CXCR3 (also known as C—X—C chemokine receptor type 3, Mig receptor, IP10 receptor, G protein). It has a coupling receptor 9, an interferon-inducible protein 10 receptor, accession number NM_001504). In some embodiments, the CD4 and / or CD8 effector memory T cells are derived from peripheral blood. In some embodiments, the CD4 and / or CD8 effector memory T cells are tumor derived.

  In some embodiments of the disclosed methods, administration of an effective amount of a human PD-1 axis binding antagonist and OX40 binding agonist to an individual is compared to prior to administration of the human PD-1 axis binding antagonist and OX40 binding agonist. , Characterized by an increase in inflammatory markers (eg CXCR3) in CD8 T cells. CXCR3 / CD8 T cells can be measured by any means known in the art and methods described in the Examples. In some embodiments, the CXCR3 / CD8 T cells are derived from peripheral blood. In some embodiments, the CXCR3 / CD8 T cells are tumor derived.

  In some embodiments of the methods of the invention, Treg function is suppressed as compared to prior to administration of the combination. In some embodiments, T cell depletion is reduced compared to before administration of the combination.

  In some embodiments, the number of Tregs is increased compared to before administration of the combination. In some embodiments, plasma interferon gamma is increased compared to before administration of the combination. The number of Tregs can be assessed, for example, by determining the percentage of CD4 + Fox3p + CD45 + cells (eg, by FACS analysis). In some embodiments, the absolute number of Tregs (eg, in the sample) is determined. In some embodiments, the Treg is from peripheral blood. In some embodiments, the Treg is derived from a tumor.

  In some embodiments, T cell priming, activation, and / or proliferation is increased compared to prior to administration of the combination. In some embodiments, the T cells are CD4 + and / or CD8 + T cells. In some embodiments, T cell proliferation is detected by determining the proportion of Ki67 + CD8 + T cells (eg, by FACS analysis). In some embodiments, T cell proliferation is detected by determining the proportion of Ki67 + CD4 + T cells (eg, by FACS analysis). In some embodiments, the T cells are derived from peripheral blood. In some embodiments, the T cell is derived from a tumor.

  Any of the PD-1 axis binding antagonists and OX40 binding agonists known in the art or described herein can be used in the methods of the present disclosure.

VI. Detection and Diagnostic Methods In some embodiments, the sample is treated prior to treatment with a PD-1 axis binding antagonist (in some embodiments, prior to treatment with an OX40 binding agonist, eg, an anti-human OX40 agonist antibody, For example, prior to combination therapy with a PD-1 axis binding antagonist). In some embodiments, the tissue sample is a formalin fixed and paraffin embedded sample, an archive sample, a fresh sample, or a frozen sample.

  In some embodiments, the sample is whole blood. In some embodiments, whole blood includes immune cells, circulating tumor cells, and any combination thereof.

  Biomarker reference levels and / or expression levels / amounts (eg, PD-L1) include, but are not limited to, DNA, mRNA, cDNA, protein, protein fragments, and / or gene copy number in the art. It can be determined qualitatively and / or quantitatively based on any known suitable criteria. In certain embodiments, the presence and / or expression level / amount of the biomarker in the first sample is increased or increased compared to the presence / absence and / or the expression level / amount in the second sample. . In certain embodiments, the presence / absence and / or expression level / amount of the biomarker in the first sample is reduced or reduced compared to the presence and / or expression level / amount in the second sample. . In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Additional disclosure for determining the presence / absence and / or expression level / amount of genes is described herein.

  In some embodiments of any of these methods, elevated expression is as described herein compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. About 10%, 20%, 30%, 40%, 50% of the level of a biomarker (eg, protein or nucleic acid (eg, gene or mRNA)) detected by standard methods known in the art such as methods %, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In certain embodiments, elevated expression refers to an increase in the expression level / amount of biomarker in the sample, which increase is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. At least about 1.5 times, 1.75 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times the expression level / amount of each biomarker in , 25 times, 50 times, 75 times, or 100 times. In some embodiments, elevated expression is about 1.5 compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (eg, a housekeeping gene). Refers to an overall increase of more than fold, about 1.75 times, about 2 times, about 2.25 times, about 2.5 times, about 2.75 times, about 3.0 times, or about 3.25 times .

  In some embodiments of any of these methods, reduced expression is as described herein compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. About 10%, 20%, 30%, 40%, 50% of the level of a biomarker (eg, protein or nucleic acid (eg, gene or mRNA)) detected by standard methods known in the art such as methods %, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In certain embodiments, reduced expression refers to a decrease in the expression level / amount of biomarker in a sample, the decrease being a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. At least about 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0 times the expression level / amount of each biomarker in .2 times, 0.1 times, 0.05 times, or 0.01 times.

  The presence and / or expression level / amount of various biomarkers in a sample can be analyzed by several methodologies, many of which are known in the art and understood by those skilled in the art, Chemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, quantitative blood-based assays (eg, serum ELISA), Biochemical enzyme activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (“PCR”), eg, quantitative real-time PCR (“qRT-PCR”) and other amplified detection Methods such as branched DNA, SISBA, TMA, etc.), RNA Includes any of a wide variety of assays that can be performed by Seq, FISH, microarray analysis, gene expression profiling, and / or continuous gene expression analysis (“SAGE”), and protein, gene, and / or tissue array analysis However, it is not limited to these. Typical protocols for assessing the status of genes and gene products are described, for example, in Ausubel et al. , Eds. , 1995, Current Protocols in Molecular Biology, Units 2 (Northern blotting), 4 (Southern blotting), 15 (immunoblotting), and 18 (PCR analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) may also be used.

  In some embodiments, the presence and / or expression level / amount of the biomarker is: (a) in a sample (such as a subject cancer sample), gene expression profiling, PCR (such as rtPCR or qRT-PCR), RNA-seq, Determined using a method comprising performing microarray analysis, SAGE, MassARRAY technique, or FISH, and b) determining the presence and / or expression level / amount of biomarkers in the sample. In some embodiments, the microarray method has or is encoded by one or more nucleic acid molecules that can hybridize under stringent conditions to a nucleic acid molecule that encodes the above gene. This involves the use of a microarray chip having one or more polypeptides (such as peptides or antibodies) that can bind to one or more of the proteins. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.

  Methods for assessing mRNA in cells are well known, such as hybridization assays using complementary DNA probes (in situ hybridization using labeled riboprobes specific for one or more genes, Northern blots, and related techniques) As well as various nucleic acid amplification assays (RT-PCR using complementary primers specific for one or more of the genes, and other amplified detection methods such as branched DNA, SISBA, TMA, etc.) Is mentioned.

  Samples from mammals can be conveniently assayed for mRNA using Northern blots, dot blots, or PCR analysis. In addition, such methods can determine the level of target mRNA in a biological sample (eg, by simultaneously testing the level of a control mRNA sequence of a “housekeeping” gene such as an actin family member) May include one or more steps. Optionally, the sequence of the amplified target cDNA can be determined.

  Optional methods include protocols that test or detect mRNA, such as target mRNA, in a tissue or cell sample by microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to produce cDNA probes. The probe is then hybridized to an array of nucleic acids immobilized on a solid support. The array is constructed so that the sequence and position of each member of the array is known. For example, various genes whose expression correlates with increased or decreased clinical benefit of anti-angiogenic therapy can be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe is derived expresses that gene.

  According to some embodiments, the presence and / or expression level / amount is measured by observing the protein expression level of the aforementioned gene. In certain embodiments, the method comprises contacting a biological sample with an antibody to a biomarker described herein (eg, an anti-PD-L1 antibody) under conditions that permit binding of the biomarker. Detecting whether a complex is formed between the antibody and the biomarker. Such methods can be in vitro methods or in vivo methods. In one embodiment, the antibodies are used to select biomarkers for selection of subjects, eg, individuals, eligible for therapy with a PD-L1 axis binding antagonist.

  In certain embodiments, the presence and / or expression level / amount of biomarker protein in a sample is tested using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method for determining or detecting the presence of proteins in a sample. In some embodiments of any of these methods, assays, and / or kits, the PD-L1 biomarker is PD-L1. In some embodiments, PD-L1 is detected by immunohistochemistry. In some embodiments, the elevated expression of the PD-L1 biomarker in the sample from the individual is elevated protein expression, and in further embodiments is determined using IHC. In one embodiment, the expression level of the biomarker comprises: (a) performing an IHC analysis of a sample (such as a target cancer sample) using an antibody; and b) determining the expression level of the biomarker in the sample. Determined using the method of inclusion. In some embodiments, the IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (eg, a control cell line stained sample or a tissue sample from a non-cancerous patient).

  IHC can be performed in combination with additional techniques such as morphological staining and / or fluorescence in situ hybridization. Two general IHC methods are available, a direct assay and an indirect assay. According to the first assay, the binding of the antibody to the target antigen is determined directly. This direct assay uses a labeling reagent such as a fluorescent tag or an enzyme-labeled primary antibody that can be visualized without further antibody interaction. In a typical indirect assay, an unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. When the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Since several secondary antibodies react with different epitopes on the primary antibody, signal amplification occurs.

Primary and / or secondary antibodies used for IHC are typically labeled with a detectable moiety. A number of labels are available and can generally be grouped into the following categories: (a) Radioisotopes, eg, ³⁵ S, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, (b ) Colloidal gold particles, (c) rare earth chelates (europium chelates), including but not limited to: Texas Red, rhodamine, fluorescein, dansyl, lysamine, umbelliferone, phycocytherin, phycocyanin, or commercially available Fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7, and / or one or more derivatives of any of the above, (d) various enzyme-substrate labels are available and are described in US Pat. No. 4,275,149. No. of these To provide an overview of several. Examples of enzyme labels include luciferases (eg, firefly luciferase and bacterial luciferase, US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidase, eg, western Horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (such as uricase and xanthine oxidase), Examples include lactoperoxidase and microperoxidase.

  Examples of enzyme and substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, alkaline phosphatase (AP) with para-nitrophenyl phosphate as a chromogenic substrate, and chromogenic substrates (eg, p-nitrophenyl-β-D-galactosidase) or β-D-galactosidase (β-D-Gal) with a fluorogenic substrate (eg 4-methylumbelliferyl-β-D-galactosidase). For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.

  In some embodiments of any of these methods, PD-L1 is detected by immunohistochemistry using an anti-PD-L1 diagnostic antibody (ie, a primary antibody). In some embodiments, the PD-L1 diagnostic antibody specifically binds to human PD-L1. In some embodiments, the PD-L1 diagnostic antibody is a non-human antibody. In some embodiments, the PD-L1 diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the PD-L1 diagnostic antibody is a monoclonal antibody. In some embodiments, the PD-L1 diagnostic antibody is directly labeled.

  The specimen prepared in this way can be placed and covered. Thereafter, the slide evaluation can be determined using, for example, a microscope and the staining intensity criteria used routinely by the art can be used. In one embodiment, when tumor-derived cells and / or tissues are examined using IHC, the staining is generally (as opposed to stroma or surrounding tissue present in the sample) tumor cells and / or Or it is understood that it is determined or evaluated in the organization. In some embodiments, when tumor-derived cells and / or tissues are examined using IHC, staining can include determination or evaluation on tumor infiltrating immune cells, such as intratumoral or peritumoral immune cells. Understood. In some embodiments, the presence of the PD-L1 biomarker is greater than 0% of the sample, at least 1% of the sample, at least 5% of the sample, or the sample by IHC, as described in Table 4 below. Of at least 10%. In some embodiments, the presence of the PD-L1 biomarker is detected by IHC in less than 5% of the cells. In some embodiments, the presence of the PD-L1 biomarker is detected by IHC in less than 1% of the cells. In some embodiments, the presence of the PD-L1 biomarker is detected by IHC in 0% of the cells.

  In some embodiments of any of these methods, assays, and / or kits, the presence of a PD-L1 biomarker is detected by IHC with any intensity PD-L1 staining. In some embodiments, the PD-L1 biomarker is detected as a weak staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected as moderate staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected as a strong staining intensity by IHC.

  In some embodiments, the PD-L1 biomarker is detected by IHC in tumor cells, tumor infiltrating immune cells, and combinations thereof.

  Anti-PD-L1 antibodies suitable for use with IHC are well known in the art. One skilled in the art will recognize that additional suitable anti-PD-L1 antibodies can be identified and characterized by comparison with anti-PD-L1 antibodies using, for example, the IHC protocols disclosed herein. to understand.

Positive tissue controls were placenta and tonsil tissue (strong PD-L1 staining intensity), HEK-293 cells transfected with recombinant human PD-L1 (weak, moderate, and strong intensity varying degrees of PD-L1 Dyeing intensity). The following may refer to exemplary PD-L1 IHC criteria.
Table 4.

  In some embodiments, PDL1 status is diagnosed according to the guidelines provided in Table 4 above.

In some embodiments, criteria for PD-L1 IHC diagnostic assessment are provided below.
Table 5

  In some embodiments, PDL1 status is diagnosed according to the guidelines provided in Table 5 above. In some embodiments, a sample having an IHC0 and / or IHC1 score may be considered negative for a PDL1 biomarker. In some embodiments, a sample having an IHC2 and / or IHC3 score may be considered positive for a PDL1 biomarker. In some embodiments, the sample is diagnosed with IHC0, IHC0 and / or 1, IHC1, IHC1 and / or 2, IHC2, IHC2 and / or 3, or IHC3.

  In some embodiments, PDL1 expression is assessed in a tumor or tumor sample. As used herein, a tumor or tumor sample can encompass some or all of the tumor area occupied by tumor cells. In some embodiments, the tumor or tumor sample further includes a tumor area occupied by tumor-associated intratumoral cells and / or tumor-associated stroma (eg, adjacent peri-tumour fibrogenic stroma). Tumor-associated intratumoral cells and / or tumor-related stroma is immediately adjacent to and / or adjacent to the main tumor mass (eg, tumor-infiltrating immune cells as described herein) The area may be included. In some embodiments, PDL1 expression is assessed in tumor cells. In some embodiments, PDL1 expression is assessed on immune cells within the tumor area described above, such as tumor infiltrating immune cells.

  In an alternative method, the sample may be contacted with an antibody specific for the biomarker under conditions sufficient to produce an antibody-biomarker complex, after which the complex is detected. The presence of the biomarker can be detected in several ways to assay a wide variety of tissues and samples such as plasma or serum, for example, by Western blotting and ELISA procedures. A wide range of immunoassay techniques using such assay formats are available, see, eg, US Pat. Nos. 4,016,043, 4,424,279, and 4,018,653. I want. These include non-competitive single site and two site or “sandwich” assays, as well as traditional competitive binding assays. These assays also involve direct binding of the labeled antibody to the target biomarker.

  The presence and / or expression level / amount of a selected biomarker in a tissue or cell sample can also be tested by functional assays or activity-based assays. For example, if the biomarker is an enzyme, assays known in the art can be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.

  In certain embodiments, samples are normalized for both differences in the amount of biomarker assayed and variations in sample quality used, as well as variations between assay runs. Such normalization can be accomplished by detecting and incorporating the expression of certain specific biomarkers, such as well-known housekeeping genes. Alternatively, normalization can be based on the average signal or median value of all of the assayed genes or a large subset thereof (global normalization approach). For each gene, the measured normalized amount of the subject tumor mRNA or protein is compared to the amount found in the reference set. The normalized expression level of each mRNA or protein per tested tumor per subject can be expressed as a percentage of the measured expression level in the reference set. The measured presence and / or expression level / amount in the particular subject sample being analyzed falls within a percentage within this range, which can be determined by methods well known in the art.

  In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary tumor or a metastatic tumor. Tissue biopsy is often used to obtain a representative piece of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissues or fluids that are known or thought to contain the tumor cells of interest. For example, lung cancer lesion samples can be obtained by excision, bronchoscopy, fine needle aspiration, bronchial abrasion, or from sputum, pleural fluid, or blood. The gene or gene product can be detected from cancer or tumor tissue, or from other body samples such as urine, sputum, serum, or plasma. The same techniques discussed above for detection of target genes or gene products in cancerous samples can be applied to other body samples. Cancer cells can shed from cancerous lesions and appear in such body samples. By screening such body samples, a simple early diagnosis of these cancers can be achieved. In addition, the progress of therapy can be more easily monitored by testing such body samples for target genes or gene products.

  In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue are from the same subject or individual obtained at one or more time points different from when the test sample was obtained. A single sample or a plurality of combined samples. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at an earlier time point than the test sample is obtained. Such a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained when the reference sample is obtained during the initial diagnosis of cancer and when the cancer becomes metastatic cancer. May be useful in some cases.

  In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple sample from one or more healthy individuals that are not subjects or individuals. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is combined from one or more individuals having a disease or disorder (eg, cancer) that is not the subject or individual. A plurality of samples. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from normal tissue, or a pool from one or more individuals that are not subjects or individuals. Plasma or serum sample. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissue, or a disease or disorder that is not a subject or individual (eg, cancer A pooled plasma or serum sample from one or more individuals having.

  In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a tumor tissue sample (eg, biopsy tissue). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is renal tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is stomach tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, and / or bone / bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is endometrial tissue. In some embodiments, the tissue sample is brain tissue (eg, glioblastoma, neuroblastoma, etc.).

  In some embodiments, a tumor tissue sample (the term “tumor sample” is used interchangeably herein) can encompass some or all of the tumor area occupied by tumor cells. In some embodiments, the tumor or tumor sample further includes a tumor area occupied by tumor-associated intratumoral cells and / or tumor-associated stroma (eg, adjacent peri-tumour fibrogenic stroma). Tumor-associated intratumoral cells and / or tumor-related stroma is immediately adjacent to and / or adjacent to the main tumor mass (eg, tumor-infiltrating immune cells as described herein) The area may be included.

  In some embodiments, tumor cell staining is expressed as a percentage of all tumor cells that exhibit any intensity of membrane staining. Infiltrating immune cell staining can be expressed as a percentage of the total tumor area occupied by immune cells exhibiting any intensity of staining. Total tumor area includes malignant cells, as well as tumor-related stroma, and includes the area of the immune infiltrate that is directly adjacent to and adjacent to the main tumor mass. In addition, infiltrating immune cell staining can be expressed as a percentage of all tumor infiltrating immune cells.

  In some embodiments of any of these methods, the disease or disorder is a tumor. In some embodiments, the tumor is a malignant cancerous tumor (ie, cancer). In some embodiments, the tumor and / or cancer is a solid tumor or a non-solid or soft tissue tumor. Examples of soft tissue tumors include leukemia (eg, chronic myelogenous leukemia, acute myeloid leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, Prolymphocytic leukemia, or hairy cell leukemia), or lymphoma (eg, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include any cancer of body tissue other than blood, bone marrow, or lymphatic system. Solid tumors can be further classified into those of epithelial cell origin and non-epithelial cell origin. Examples of solid epithelial cell tumors include the gastrointestinal tract, colon, colorectal (eg, basal colorectal cancer), breast, prostate, lung, kidney, liver, pancreas, ovary (eg, endometrioid ovarian cancer), head and neck Part, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, lips, nasopharynx, skin, uterus, male genital organs, urinary organs (eg, urothelial cancer, atypical urothelial cancer, transitional cell carcinoma), bladder And skin tumors. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is secondary or tertiary locally advanced or metastatic non-small cell lung cancer. In some embodiments, the cancer is an adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (eg, triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or Hepatocellular carcinoma. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor at a second site from any of the above cancer types.

  In some embodiments of any of these methods, the cancer presents human effector cells (eg, infiltrated by human effector cells). For example, methods for detecting human effector cells, such as a method using IHC, are well known in the art. In some embodiments, the cancer exhibits high levels of human effector cells. In some embodiments, the human effector cell is one or more of NK cells, macrophages, monocytes. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (eg, triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or Hepatocellular carcinoma.

  In some embodiments of any of these methods, the cancer presents cells that express FcR (eg, infiltrated by cells that express FcR). For example, FcR detection methods such as the IHC method are well known in the art. In some embodiments, the cancer presents cells that express high levels of FcR. In some embodiments, the FcR is FcγR. In some embodiments, the FcR is an activated FcγR. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (eg, triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or Hepatocellular carcinoma.

  In some embodiments, the PD-L1 biomarker is a FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot method, immunodetection method, HPLC, surface plasmon resonance, optical spectroscopy Using a method selected from the group consisting of optics, mass spectroscopy, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques, and FISH, and combinations thereof Detected in the sample. In some embodiments, the PD-L1 biomarker is detected using FACS analysis. In some embodiments, the PD-L1 biomarker is PD-L1. In some embodiments, PD-L1 expression is detected in a blood sample. In some embodiments, PD-L1 expression is detected in circulating immune cells in a blood sample. In some embodiments, the circulating immune cells are CD3 + / CD8 + T cells. In some embodiments, immune cells are isolated from a blood sample prior to analysis. Any suitable isolation / concentration method of such cell populations can be used, including but not limited to cell sorting. In some embodiments, PD-L1 expression is elevated in a sample from an individual that responds to treatment with a PD-L1 / PD-1 axis pathway inhibitor, such as an anti-PD-L1 antibody. In some embodiments, PD-L1 expression is elevated in circulating immune cells such as CD3 + / CD8 + T cells in the blood sample.

  Expression level of one or more marker gene, protein (s) (eg, cytokine, eg, gamma interferon), and / or cell composition in a sample comprising leukocytes obtained from a subject (eg, Treg percentage and Measuring the absolute number of Tregs, eg, the number of CD8 + effector T cells) (the subject has been treated with a PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) One or more marker genes were selected from a T cell marker gene, or a memory T cell marker gene (eg, a marker for effector memory T cells), and this treatment was obtained from a subject compared to a reference. One or more marker genes, protein (s) in the sample, and / or Determine pharmacodynamic activity based on expression level of cell composition (increased expression level of one or more marker genes compared to baseline indicates pharmacodynamic activity for OX40 agonist treatment) A method for monitoring pharmacodynamic activity of OX40 agonist treatment is provided herein. The expression level of the marker gene, protein, and / or cell composition is measured by one or more methods described herein.

  As used herein, “pharmacodynamic (PD) activity” can refer to the effect of treatment on a subject (eg, a combination treatment of an OX40 agonist and a PD-1 axis antagonist). An example of PD activity may include modulation of the expression level of one or more genes. Without wishing to be bound by theory, it is believed that monitoring PD activity, such as by measuring the expression of genetic markers, may be advantageous during clinical trials testing OX40 agonists and PD-1 axis antagonists. PD activity monitoring can be used, for example, to monitor response to therapy, toxicity, and the like.

  In some embodiments, the level of expression of one or more marker genes, proteins, and / or cell compositions has not been treated (eg, a combination treatment with an OX40 agonist and a PD-1 axis binding antagonist) It can be compared to a standard that can include a sample of origin. In some embodiments, the criteria can include a sample from the same subject prior to receiving treatment (eg, a combination treatment of an OX40 agonist and a PD-1 axis binding antagonist). In some embodiments, the reference may include a reference value for one or more samples from other subjects undergoing treatment (eg, a combination treatment of an OX40 agonist and a PD-1 axis antagonist). For example, a patient population can be treated and an average, average, or median expression level of one or more genes can be generated from that population as a whole. Sample sets obtained from cancers having common characteristics (eg, exposure to a common treatment such as the same cancer type and / or stage, or a combination treatment of an OX40 agonist and a PD-1 axis binding antagonist) are clinical Can be studied from a population such as an outcome study. This set can be used to derive a reference, for example a reference number, to which the sample of interest can be compared. Any of the criteria described herein can be used as a criteria for monitoring PD activity.

  Expression levels of one or more marker gene, protein (s) (eg, cytokines, eg, gamma interferon), and / or cell composition in a sample in leukocytes obtained from a subject (eg, Treg percentage and Measuring the absolute number of Tregs, eg, the number of CD8 + effector T cells in a peripheral blood sample (the subject is treated with a PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody)) One or more marker genes are selected from a T cell marker gene or a memory T cell marker gene (eg, an effector memory T cell marker) One or more marker genes, protein (s) in a sample obtained from Classifying as responsive or non-responsive to this treatment based on the expression level of the cell composition (increased expression level of one or more marker genes compared to the baseline is a response to OX40 agonist treatment) Provided herein is a method of monitoring a subject's responsiveness to OX40 agonist treatment. The expression level of the marker gene, protein, and / or cell composition is measured by one or more methods described herein.

  In some embodiments, criteria for monitoring responsiveness may include a sample from a subject not receiving treatment (eg, a combination treatment with an OX40 agonist and a PD-1 axis binding antagonist). In some embodiments, the criteria for monitoring responsiveness may include a sample from the same subject prior to receiving treatment (eg, a combination treatment of an OX40 agonist and a PD-1 axis binding antagonist). In some embodiments, the criteria for monitoring responsiveness is that of one or more samples from other patients undergoing treatment (eg, combination treatment with an OX40 agonist and a PD-1 axis binding antagonist). A reference value may be included. For example, a patient population can be treated and an average, average, or median expression level of one or more genes can be generated from that population as a whole. Sample sets obtained from cancers having common characteristics (eg, exposure to a common treatment such as the same cancer type and / or stage, or OX40 agonist) can be studied from a population such as a clinical outcome study. This set can be used to derive a reference, for example a reference number, to which the sample of interest can be compared. Any of the criteria described herein can be used as a criteria for monitoring PD activity.

  Certain aspects of the present disclosure relate to measuring the expression level of one or more genes or one or more proteins in a sample. In some embodiments, the sample can include white blood cells. In some embodiments, the sample can be a peripheral blood sample (eg, from a patient with a tumor). In some embodiments, the sample is a tumor sample. Tumor samples can include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basement membrane, and any other cell type associated with the tumor. In some embodiments, the sample is a tumor tissue sample containing tumor infiltrating leukocytes. In some embodiments, the sample can be processed to separate or isolate into one or more cell types (eg, white blood cells). In some embodiments, the sample can be used without separating or isolating cell types.

  A tumor sample can be obtained from a subject by any method known in the art including, but not limited to, biopsy, endoscopy, or surgical procedure. In some embodiments, tumor samples can be prepared by methods such as freezing, fixation (eg, using formalin or similar fixatives), and / or embedding with paraffin wax. In some embodiments, a tumor sample can be sectioned. In some embodiments, fresh tumor samples (ie, tumor samples that have not been prepared by the methods described above) can be used. In some embodiments, tumor samples can be prepared by incubation in solution to preserve mRNA and / or protein integrity.

  In some embodiments, the sample can be a peripheral blood sample. Peripheral blood samples can include leukocytes, PBMC, and the like. Any technique for isolating leukocytes known in the art from peripheral blood samples can be used. For example, a blood sample may be taken, red blood cells may be lysed, and white blood cell pellets may be isolated and used for the sample. In another example, density gradient separation can be used to separate white blood cells (eg, PBMC) from red blood cells. In some embodiments, fresh peripheral blood samples (ie, peripheral blood samples not prepared by the methods described above) can be used. In some embodiments, peripheral blood samples can be prepared by incubation in solution to preserve mRNA and / or protein integrity.

  In some embodiments, responsiveness to treatment results in prolonged survival (including overall survival and progression-free survival), objective response (including full or partial response). Or any one or more of the improvement of signs or symptoms of cancer. In some embodiments, responsiveness refers to an improvement in one or more factors according to a published set of RECIST guidelines for determining tumor status, ie, response, stability, or progression in cancer patients. obtain. For a more detailed discussion of these guidelines, see Eisenhauer et al. , Eur J Cancer 2009; 45: 228-47, Topalian et al. , N Engl J Med 2012; 366: 2443-54, Wolkok et al. , Clin Can Res 2009; 15: 7412-20, and Therase, P .; , Et al. J. et al. Natl. Cancer Inst. 92: 205-16 (2000). A responsive subject may refer to a subject whose cancer (s) exhibit an improvement according to one or more factors based on, for example, RECIST criteria. A non-responsive subject may refer to a subject whose cancer (s) do not show improvement according to one or more factors based on, for example, RECIST criteria.

  Conventional response criteria may not be appropriate to characterize the anti-tumor activity of an immunotherapeutic agent and may result in a delayed response that may be preceded by an initial apparent radiological progression, such as the appearance of a new lesion. Therefore, a modified response criterion has been developed that accounts for the possibility of the appearance of new lesions and allows for radiological progression that is confirmed in subsequent assessments. Thus, in some embodiments, responsiveness may refer to an improvement in one of more factors that comply with immune-related response criteria 2 (irRC). See, for example, Wolchok et al. Clin Can Res 2009; 15: 7412-20. In some embodiments, new lesions are added to the defined tumor burden, eg, followed by radiological progression at subsequent assessments. In some embodiments, the presence of non-target lesions is included in the assessment of complete response and not in the assessment of radiological progression. In some embodiments, radiological progression can be determined based solely on measurable disease and / or can be confirmed by a continuous assessment of 4 weeks or more from the first recorded date.

  In some embodiments, responsiveness can include immune activation. In some embodiments, responsiveness can include therapeutic efficacy. In some embodiments, responsiveness can include immune activation and therapeutic efficacy.

VI. Product or Kit In another embodiment of the invention, a product or kit comprising a PD-1 axis binding antagonist and / or an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) is provided. In some embodiments, the product or kit uses a PD-1 axis binding antagonist in combination with an OX40 binding agonist to treat or delay the progression of cancer in an individual, or for an individual with cancer. It further includes a package insert containing instructions for enhancing immune function. Any of the PD-1 axis binding antagonists and / or OX40 binding agonists described herein can be included in a product or kit.

  In some embodiments, the PD-1 axis binding antagonist and OX40 binding agonist (eg, an anti-human OX40 agonist antibody) are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on the container or a label associated with the container may indicate directions for use. The product or kit may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the product further comprises one or more of another agent (eg, a chemotherapeutic agent and an antineoplastic agent). Suitable containers for one or more medicaments include, for example, bottles, vials, bags, and syringes.

  This specification is considered to be sufficient to enable one skilled in the art to practice the invention. In addition to the modifications shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description, which fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

  The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. The examples and embodiments described herein are for illustrative purposes only, and various modifications or alterations have been proposed to those skilled in the art that take into account the spirit and scope of this specification and the accompanying It is understood that it should be included within the scope of the claims.

Materials and Methods In vivo tumor models: CT26 and MC38 colorectal cell lines were maintained at Genentech. For the CT26 study, 100,000 CT26 cells were inoculated subcutaneously into the right flank of 8-10 week old female Balb / c mice (Charles River Laboratories, Hollister, Calif.). For the MC38 study, 8-10 week old female C57BL / 6 mice (Charles River Laboratories) were inoculated subcutaneously with 100,000 MC38 cells in the right flank. When tumors reached an average tumor volume of approximately 150 mm 3, mice were mobilized and randomized into treatment groups, and antibody treatment started on the first day of the next day. All animal studies were performed according to the guidelines and rules described in the Animals Welfare Act and The Guide for the Care and Use of Laboratory Animals and IACUC Guidelines. The treatment groups were as follows: 1) Control antibody, initial dose of 10 mg / kg (intravenous), then 5 mg / kg (intraperitoneal), BIW × 2, n = 5, 2) Mouse anti-mouse OX40 Agonist monoclonal antibody (chimeric antibody with rat anti-mouse OX40 variable region derived from OX86 antibody and mouse IgG2a Fc. Therefore, this mouse antibody has the ability of effector function including, but not limited to ADCC), first dose 0 .1 mg / kg (intravenous), then intraperitoneal, TIW × 2, n = 5, 3) Mouse anti-PD-L1, initial dose 10 mg / kg (intravenous), then 5 mg / kg (intraperitoneal), TIW × 2, n = 5, and 4) Mouse anti-mouse OX40 monoclonal antibody, initial dose 0.1 mg / kg (intravenous), then intraperitoneally, T IW × 2 and mouse anti-PD-L1, initial dose 10 mg / kg (intravenous), then 5 mg / kg (intraperitoneal) TIW × 2, n = 5. Mice were sacrificed and peripheral blood was collected 9 days after the first dose.

  Antibodies: All treatment antibodies were generated at Genentech. The control antibody was anti-gp120 mouse IgG1, clone 10E7.1D2. The anti-OX40 antibody was clone OX-86 mouse IgG2a (generated by cloning the rat anti-mouse OX40 agonist antibody OX-86 into the mouse IgG2a backbone) and anti-PDL1 was clone 6E11.1.9 mouse IgG1 . The dosing schedule of initial dosing by intravenous (IV) or single dosing followed by intraperitoneal (IP) administration is shown in the figure legend. The antibody was diluted in either PBS or 20 mM histidine acetate, 240 mM sucrose, 0.02% polysorbate 20 (pH 5.5). TIW indicates dosing 3 times a week and BIW indicates dosing twice a week.

  Tumor treatment and flow cytometry: After tumor removal and chopping with a razor blade, 5% fetal calf serum and Collagenase D (Roche, Indianapolis, on a rocking platform in C-tube (Miltenyi Biotec, San Diego, Calif.)) IN) (0.25 mg / mL) as well as in RPMI-1640 medium with DNAse I (Roche) (0.1 mg / mL) for 15 minutes at 37C. After incubation, the tumors were processed on gentleMACS (Miltenyi Biotec), filtered and washed to obtain a single cell suspension. Cells were counted with a Vi-Cell counter (Beckman Coulter, Brea, CA).

  Peripheral blood was evaluated for T cell activation and proliferation by flow cytometry. 50 uL of blood was stained with commercial antibodies against CD45, CD3, CD4, CD8, CXCR3 (all BD Biosciences) and Ki67 (eBiosciences) according to the manufacturer's instructions. Initially, cells were stained with Live / Dead Near-Infrared viability dye (Life Technologies, Grand Island, NY) in PBS on ice for 30 minutes and then washed. The cells were then blocked with purified anti-CD16 / −CD32 (BD Biosciences, San Jose, Calif.) And then surface stained in PBS + 0.5% BSA + 2 mM EDTA buffer on ice for 30 minutes. To assess OX40 expression of PD-1 and T cells, cells were stained as follows: PD1-FITC, CD3-PerCp. Cy5.5, CD4-PE-Cy7, CD8 Pacific Blue, CD45 v500 (BD Biosciences), OX40 Alexa Fluor 647 (Genentech, clone 1H1). To assess PDL1 expression in various cell types, staining was performed as follows: CD11b-FITC, Gr-1 PE-Cy7, CD8 Alexa 700, CD45 v500, CD4-PerCp. Cy5.5 (BD Biosciences), PDL1-biotin (Genentech, clone 6F8.2.5) followed by streptavidin-PE (BD Bioscience). To evaluate Foxp3 + regulatory T cell populations, the cell surface was first stained with CD45-PE-Cy7, CD4 PerCp-Cy5.5 (BD Biosciences), and then 1 × foxp3 fixation / permeabilization buffer ( eBioscience, San Diego, Calif.) overnight at 4C. The cells were then permeabilized in 1 × foxp3 permeabilization buffer (eBioscience) and stained with Foxp3-FITC (eBioscience). Stained cells were obtained using Fortessa's FACS Diva software or FACS Canto II (BD Biosciences) and then analyzed with FlowJo software.

  Tumor dissection and Fluidigm expression analysis: RNA was extracted from UBC or NSCLC FFPE-derived archive tumors as described (Poles, T., et al. (2014) Nature 515: 558-62, Herbst, R., et al. S., et al (2014) Nature 515: 563-7). Briefly, tumor FFPE sections were macrodissected to concentrate neoplastic tissue, and the tissue was lysed using tumor lysis buffer and proteinase K to allow complete digestion and nucleic acid release. RNA was isolated using the High Pure FFPE RNA micro kit (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer's protocol.

Gene expression analysis was performed using the BioMark HD real-time PCR platform (Fluidigm) as previously described (Shames, DS, et al. (2013) PLoS ONE 8: e56765). All Taq-man assays in the expression panel are FAM-MGB, ordered from Life Technologies, either as a build-to-order or custom designed, and included four reference genes: SP2, GUSB, TMEM55B, and VPS33B. . The geometric median of C _t values for these four reference genes (SP2, GUSB, TMEM55B, and VPS33B) is calculated for each sample and the expression level using the delta C _t (DC _t ) method as follows: Was determined: C _t (target gene) 2 geometric median C _t (reference gene). The median mRNA expression level across the study patients (measured by immune chip (iChip)) was used as a cutoff to derive high expression versus low expression categorization. P value was determined by t-test.

  PD-L1 immunohistochemistry (IHC): Formalin-fixed paraffin-embedded (FFPE) sections of tumor samples or cancer cell lines were analyzed.

  After deparaffinization of formalin-fixed paraffin-embedded tissue sections, the antigen was collected, blocked and incubated with primary anti-PD-L1 antibody. After incubation with secondary antibody and enzyme color development, sections were counterstained, dehydrated with a series of alcohols and xylene, and then covered.

The following protocol was used for IHC. Stain 4 mm thick formalin-fixed paraffin-embedded (FFPE) tissue sections against PD-L1 with anti-human PD-L1 rabbit monoclonal antibody on an automated staining platform using a concentration of 4.3 mg / mL Signals were visualized with diaminobenzidine and sections were counterstained with hematoxylin. The following scoring scheme was used to assess PD-L1 expression on tumor infiltrating immune cells.

PD-L1 IHC staining was performed using the following reagents and materials using a Ventana Benchmark XT or Benchmark Ultra system.
Primary antibody: anti-PD-L1 rabbit monoclonal primary antibody Specimen type: formalin-fixed paraffin-embedded (FFPE) sections of tissue samples of various staining intensities and control cell pellets Species used for procedure: human Instrument: BenchMark XT or Benchmark Ultra
Epitope recovery conditions: cell conditioning, standard 1 (CC1, Ventana, catalog number 950-124)
Primary antibody conditions: 1/100, 6.5 μg / mL / 36 ° C. for 16 minutes Diluent: antibody dilution buffer (Tris-buffered saline containing carrier protein and Brig-35)
Negative control: 6.5 μg / mL naive rabbit IgG (Cell Signaling) or diluent only Detection: Optiview or Ultraview Universal DAB detection kit (Ventana), and amplification kit (if applicable) according to manufacturer's instructions (Ventana) used.
Counterstaining: Ventana Hematoxylin II (Catalog Number 790-2208) / Bluing Reagent (Catalog Number 760-2037) (4 minutes and 4 minutes, respectively)

The benchmark protocol was as follows.
1. Paraffin (selection)
2. Deparaffinization (selection)
3. Cell conditioning (selection)
4). Conditioning device number 1 (selection)
5). Standard CC1 (selection)
6). Ab incubation temperature (selection)
7.36C Ab Inc. (Choice)
8). Titration (selection)
9. Automatic dispensing (primary antibody) and incubation (16 minutes)
10. Counterstaining (selection)
11. Application of a drop of (hematoxylin II) (counter stain), application of coverslip, incubation (4 minutes)
12 After counterstaining (selection)
13. Application of a drop of (BLUING reagent) (after counterstaining), application of coverslips, incubation (4 minutes)
14 15. Wash the slide with soapy water to remove the oil. 15. Rinse slide with water Slides are dehydrated with 95% ethanol, 100% ethanol and xylene (Leica automatic dyeing machine program number 9)
17. Cover with slip

Results It is known that OX40 is a costimulatory molecule expressed in activated CD4 T cells (Teff) and T regulatory (Treg) cells. OX40 is not constitutively expressed on naive T cells, but is induced after involvement of the T cell receptor (TCR). It is known that ligation of OX40 in the presence of TCR stimulation enhances Teff cell activation and enhances effector T cell function by a dual mechanism that inhibits Treg cells. It was found that anti-OX40 treatment reduces Treg activity in an in vitro Treg suppression assay. These results demonstrate that OX40 agonist treatment can modulate several critical T cell functions.

  Inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity for the treatment of cancer (eg, tumor immunity) and infections, eg, both acute and chronic (eg, persistent) infections. ing.

  We tested whether intratumoral T cells express PD-1 and OX40. As shown in FIG. 1, intratumoral CD8 + T cells expressed inhibitory receptors such as PD-1, but most of these cells also expressed OX40. This result suggests that OX40 stimulation of effector T cells can counteract the effects of PD-1 and other inhibitory receptors expressed on T cells.

  Treatment with anti-OX40 agonist antibody (single agent) significantly reduced the ratio of intratumoral Foxp3 + regulatory T cells to the total number of CD45 + cells (CD45 defines all hematopoietic cells such as leukocytes, FIG. 2A) The absolute number of Foxp3 + Treg was also significantly reduced (FIG. 2B). In addition, combined treatment with anti-OX40 agonist antibody and anti-PDL1 antagonist antibody significantly reduced the ratio of intratumoral Foxp3 + regulatory T cells to the total number of CD45 + cells (FIG. 2A), and also significantly reduced the absolute number of intratumoral Foxp3 + Tregs. (FIG. 2B). These results demonstrated that OX40 agonist-mediated reduction of intratumoral Foxp3 + Treg is maintained when OX40 agonist is administered in combination with an anti-PDL1 antagonist.

  We tested the effect of OX40 agonist treatment on PD-L1 expression. Treatment with an anti-OX40 agonist significantly reduces PD-L1 expression in tumor cells and intratumoral myeloid cells, suggesting that PD-L1 may limit anti-OX40 efficacy in a negative feedback manner (FIG. 3A). And B). Without being bound by theory, these results suggest that treatment with an OX40 agonist may enhance treatment with a PD-1 axis inhibitor because treatment with an OX40 agonist increased PD-L1 expression. Is done. Clinical data correlates increased PDL1 expression as an expanded response to PD1 axis antagonists (eg, anti-PD-L1 antagonist antibodies).

  Treatment with anti-OX40 agonist antibody and anti-PDL1 antagonist antibody demonstrated synergistic combined efficacy in CT26 and MC38 colorectal cancer syngeneic tumor models (FIGS. 4A and B, 5A and B). Analysis of individual tumor volume measurements (individual mice in each experiment, FIGS. 4B, 5B) showed that animals treated with the combination compared to animals treated with either agent (OX40 agonist, PDL1 antagonist) alone, It became clear that it showed a significant reduction in tumor size more frequently. In other words, the frequency of animals showing partial and complete responses is significantly higher in animals treated with the combination compared to animals treated with either drug alone.

  Analysis of peripheral blood collected from co-treated CT26 mice revealed an increase in effector cell proliferation and inflammatory T cell markers (FIGS. 9A, B, C, and D). The proliferation levels of CD8 + T cells (FIG. 9A), Treg cells (FIG. 9B), plasma interferon gamma levels (FIG. 9C), and activated T cells (FIG. 9D) were tested. Increased proliferation (Ki67), plasma interferon gamma, and inflammatory markers (Tbet, CXCR3) in the combined arm (compared to either single agent arm) aPD1 (checkpoint blockade) and aOX40 (costimulatory) activity Synergy is revealed.

  Specifically, proliferating CD8 + T cell levels (expressed as a percentage of ki67 + / total CD8 + T cells) are compared to treatment with OX40 agonist or PDL1 antagonist alone in animals treated with OX40 agonist and PD-L1 antagonist alone. There was a marked increase (Figure 9A). Proliferating CD8 + T cell levels in animals treated in combination are higher than the additive effect of the single agent treated population, and a synergistic effect of combining OX40 agonist treatment and PD-1 axis inhibition was detected by analysis of peripheral blood markers and cells. Demonstrate that you get.

  In addition, a reduction in peripheral blood Treg was observed with treatment with OX40 agonist alone, and a reduction in peripheral blood Treg was maintained in the combined (OX40 agonist and PDL1 antagonist) therapy arm (FIG. 9B). An increase in plasma gamma interferon was observed with the combination of OX40 agonist and PDL1 antagonist (FIG. 9C).

  The chemokine receptor CXCR3 is a Gαi protein coupling receptor of the CXC chemokine receptor family. There are two variants of CXCR3, CXCR3-A binds to CXC chemokines CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC), while CXCR3-B is CXCL9, In addition to CXCL10 and CXCL11, it can also bind to CXCL4 (Clark-Lewis, I., et al. (2003) J. Biol. Chem. 278 (1): 289-95). CXCR3 is mainly expressed on activated T lymphocytes and NK cells, as well as some epithelial cells. CXCR3 and CCR5 are preferentially expressed in Th1 cells and are upregulated in effector memory CD8 T cells (Goom, JR and Luster, AD (2011) Exp. Cell Res. 317 (5). : 620-31). CXCR3 can control leukocyte trafficking. Chemokine binding to CXCR3 induces a variety of cellular responses, most notably inflammatory cell integrin activation, cytoskeletal changes, and chemotactic migration (Groom, JR and Luster, AD (2011) Exp. Cell Res. 317 (5): 620-31).

  Activated T cell levels (specifically determined using activated memory Teff cells, CXCR3 marker) are compared with treatment with OX40 agonist or PDL1 antagonist alone with OX40 agonist and PD-L1 antagonist. There was a marked increase in animals treated with the combination (FIG. 9D). The level of memory effector T cells (CXCR3 +) in animals treated in combination is higher than the additive effect of the single agent treated population, and the synergistic effect of combining OX40 agonist treatment and PD-1 axis inhibition is a peripheral blood marker and cell It can be detected by analysis of Increased proliferation (Ki67) and inflammatory marker (CXCR3) on CD8 T cells in the combined treatment arm suggests enhanced cytotoxicity through synergy of anti-PDL1 (checkpoint blocking) and anti-OX40 (costimulatory) activities obtain.

  In addition, the combined treatment effect is detected by an increase in effector and inflammatory T cell markers (eg, by rtPCR (Fluidigm)) analyzed in tumor samples treated in combination versus samples treated with either agent alone. did. For example, markers of Treg (Fox3p), CD8 + Teff (CD8b), and activated T cells (eg, Tbet, CXCR3, eg, interferon gamma response related genes) can be analyzed.

  Experiments were conducted to test the dose response effect of OX40 agonist antibody treatment in a CT26 colorectal cancer syngeneic tumor model. Anti-OX40 agonist antibody single agent treatment showed dose response (Figure 6A, B). The 0.1 mg / mL dose showed submaximal efficacy and was selected for further combination treatment experiments.

  FIGS. 7A and B show the results of a combination treatment of a sub-therapeutic dose of an anti-OX40 agonist antibody and an anti-PDL1 antagonist antibody compared to treatment with either agent alone. Synergistic combination efficacy is observed, suggesting that the maximum effective dose of OX40 agonist antibody may be lower than when combined with a PD-1 axis antagonist.

  FIGS. 8A and B show the results of a combined treatment of a sub-therapeutic level of anti-OX40 agonist antibody and an anti-PDL1 antagonist antibody as compared to treatment with either agent alone. . Synergistic combination efficacy is observed, suggesting that the maximum effective dose of the OX40 agonist antibody may be lower than when the OX40 agonist antibody is provided in combination with a PD-1 axis antagonist.

  FIG. 10 shows the relationship between OX40 expression and PDL1 diagnostic status in cancer samples from human patients with urothelial bladder cancer (UBC) and non-small cell lung cancer (NSCLC). The tissue sample was from a patient who participated in a phase 1 clinical trial using the anti-PD-L1 antibody MPDL3280A. The IHC disclosed herein was used to determine the PD-L1 biomarker status of tumor infiltrating immune cells (IC). The OX40 expression level was determined using rtPCR analysis (Fluidigm). In UBC, OX40 expression was observed in patients with 0 or 1 PDL1 IHC status. The level of OX40 expression correlated with PDL1 IHC status, and increased PDL1 expression correlated with increased OX40 expression. In NSCLC, OX40 expression was observed by IHC in patients with low or no PDL1 expression (and samples with 2 and 3 PDL1 IHC status). These results may (a) improve response to combined treatment of PD-1 axis binding antagonists and OX40 binding agonists (eg, anti-human OX40 agonist antibodies) in patients with PDL1 IHC0 and / or 1 status. (B) the potential for improved response to a combination treatment of a PD-1 axis binding antagonist and an OX40 binding agonist (eg, an anti-human OX40 agonist antibody) in a patient who does not respond to PD-1 axis binding antagonist pretreatment; and (C) The potential for improved response to combination treatment of PD-1 axis binding antagonists and OX40 binding agonists (eg, anti-human OX40 agonist antibodies) in patients with PDL1 IHC2 and / or 3 conditions is suggested.

  Results of clinical studies evaluating the anti-PD-L1 antibody MPDL3280A for use in cancer therapy suggested that PD-L1 expression is associated with a clinical response to MPDL3280A. It was found that the association between PDL1 expression on tumor infiltrating immune cells and treatment response appeared to be stronger than the association with PDL1 expression on tumor cells. Tumor infiltrating immune cells may be sensitive to IFNg expression and may preferentially play a role in suppressing existing T cell responses prior to treatment (Herbst, RS, et al (2014) Nature 515: 563-). 7). Without wishing to be bound by theory, OX40 agonist treatment increases IFNg expression, resulting in enhanced PDL1 expression in tumor infiltrating immune cells and a concomitant increase in responsiveness to PD-1 axis binding antagonist treatment. It is thought to get. Thus, a combination treatment comprising an OX40 binding agonist and a PD-1 axis binding antagonist may be useful for the treatment of patients with lower PDL1 biomarker status.

  Scoring of PD-L1 expression by IHC: Using an anti-PD-L1 specific antibody capable of detecting PD-L1 in human formalin-fixed paraffin-embedded (FFPE) tissue by IHC, PD- in tumor specimens The presence or absence of L1 expression was evaluated. In order to measure and quantify the relative expression of PD-L1 in tumor samples, a PD-L1 IHC scoring system was developed to measure PD-L1 specific signals in tumor cells and tumor infiltrating immune cells. Immune cells are defined as cells with lymphocyte and / or macrophage / histosphere morphology.

  Tumor cell staining is expressed as the percentage of all tumor cells exhibiting any intensity of membrane staining. Infiltrating immune cell staining is defined as the percentage of the total tumor area occupied by immune cells exhibiting any intensity of staining. Total tumor area includes malignant cells, as well as tumor-related stroma, and includes the area of the immune infiltrate that is directly adjacent to and adjacent to the main tumor mass. In addition, infiltrating immune cell staining is defined as the percentage of all tumor infiltrating immune cells.

  There was a wide dynamic range of PD-L1 staining intensity in the tumor tissue. Regardless of subcellular localization, signals were also classified as strong, moderate, weak, or negative staining.

  As shown in FIG. 11, the negative signal intensity was characterized by the absence of any detectable signal, as illustrated using HEK-293 cells (FIG. 11A). In contrast, positive signal intensity was characterized by golden to dark brown membrane staining, as illustrated using HEK-293 cells transfected with recombinant human PD-L1 (FIGS. 11B-D). checking). Finally, membrane patterns characterized by positive signal intensity, staining of placental trophoblast cells (FIG. 11E), and intense staining in the tonsillar fossa region (FIG. 11F), and often golden to dark brown staining Also illustrated by. In tumor tissue, PD-L1 negative samples were quantified as having no detectable signal or only having weak cytoplasmic background staining when assessed using a 20 × objective. In contrast, PD-L1 positive samples showed mainly membrane staining in tumor cells and / or infiltrating immune cells. PD-L1 staining was observed at various intensities ranging from weak (light brown thin film) to strong (dark brown thick film) that are easily recognized even at low magnification.

  Three representative PD-L1 positive tumor samples are shown in FIG. In the case of triple negative breast cancer, it was observed that most tumor cells were highly positive for PD-L1 and showed a combination of membrane staining and cytoplasmic staining (100 magnification) (FIG. 12A). In the case of malignant melanoma, immune cell clusters were observed, membrane staining for some of them was observed for PD-L1, and membrane staining for PD-L1 for rare tumor cells (arrows) was observed (400 magnification) (FIG. 12B). In the case of NSCLC, adenocarcinoma, immune cell clusters with strong staining for PD-L1 were observed, and several tumor cells (arrows) with membrane staining and / or cytoplasmic staining for PD-L1 were observed (400 magnification) (FIG. 12C).

  The staining in the positive case tended to be the focus distribution and intensity versus the spatial distribution and intensity. PD-L1 status was determined using a visual estimate and use of the percentage of tumors or immune cells that show any intensity staining. An isotype negative control was used to assess the presence of background in the test sample.

  Staining required a first continuous tissue section of H & E, a second continuous tissue section of anti-PD-L1, and a third continuous tissue section of an isotype negative control. A PD-L1 transfected HEK-293 cell line control or tonsil slide was used as a running control and reference for assay specificity.

PDL-1 status criteria

  The table shown above describes one embodiment for determining PDL1 status using PDL1 staining criteria. In another embodiment, a sample with an IHC score of IHC 0 and / or 1 can be considered PDL1 negative, while a sample with an IHC score of IHC 2 and / or 3 can be considered PDL1 positive. In some embodiments, PDL1 expression (eg, PDL1 staining) on the tumor itself is assessed.

  In some cases, PD-L1 positive status is either tumor cells or tumor infiltrating immune cells in up to 50% of the tumor area occupied by tumor cells, associated intratumoral, and adjacent peritumoral fibrogenic stroma. May include the presence of any intensity distinguishable PD-L1 staining. Thus, PD-L1 positive staining comprises up to 50% of tumor cells or tumor infiltrating immune cells that display any intensity staining.

  Evaluable slides stained with anti-PD-L1 were evaluated as described above. Negative staining intensity was characterized by any detectable signal or absence of signal and absence of membrane enhancement characterized by a light gray to blue color (not brown or brown). Negative if no membrane staining is present (eg, absent).

  Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (124)

  A method of treating cancer or a method of delaying its progression in an individual, said method comprising administering to said individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist, wherein said individual has cancer Or the method wherein the cancer cells in the tumor sample of the cancer from which the individual has been diagnosed do not express PD-L1.   The method of claim 1, wherein the PD-L1 biomarker is not present in the sample when contained in 0% of the sample.   3. The method of claim 2, wherein the PD-L1 biomarker is determined by protein expression measured by an immunohistochemistry (IHC) method.   A method of treating cancer or a method of delaying its progression in an individual, said method comprising administering to said individual an effective amount of a human PD-1 axis binding antagonist and an OX40 binding agonist, wherein said individual has cancer Or the method wherein the cancer cells in the tumor sample of the cancer derived from the individual express PD-L1.   The method of claim 4, wherein the PD-L1 biomarker is present in the sample when contained in greater than 0% of the sample.   6. The method of claim 4 or claim 5, wherein the PD-L1 biomarker is detected in 0% to 1% of the sample.   6. The method of claim 4 or claim 5, wherein the PD-L1 biomarker is detected in 0% to 5% of the sample.   8. The method according to any one of claims 5 to 7, wherein the PD-L1 biomarker is detected in the sample by protein expression as determined by immunohistochemistry (IHC) methods.   The PD-L1 biomarker is detected using an anti-PDL1 antibody, and the PD-L1 biomarker is detected as a weak staining intensity by IHC, a moderate staining intensity by IHC, or a strong staining intensity by IHC 9. The method of claim 8, wherein:   9. The PD-L1 biomarker is detected using an anti-PDL1 antibody, and the PD-L1 biomarker is detected as a moderate staining intensity by IHC or as a strong staining intensity by IHC. The method described.   11. The method according to any one of claims 8 to 10, wherein the sample has an IHC score of IHC0 or IHC1.   12. The method according to any one of claims 1 to 11, wherein the individual has a cancer that is resistant to a PD-1 axis binding antagonist.   13. The method of any one of claims 1-12, wherein the individual is refractory to a PD-1 axis binding antagonist.   14. The method of any one of claims 1 to 13, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PDL1 binding antagonist, and a PDL2 binding antagonist.   15. The method of claim 14, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.   16. The method of claim 15, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner.   16. The method of claim 15, wherein the PD-1 binding antagonist inhibits PD-1 binding to PDL1.   16. The method of claim 15, wherein the PD-1 binding antagonist inhibits PD-1 binding to PDL2.   16. The method of claim 15, wherein the PD-1 binding antagonist inhibits PD-1 binding to both PDL1 and PDL2.   20. The method according to any one of claims 15-19, wherein the PD-1 binding antagonist is an antibody.   16. The method of claim 15, wherein the PD-1 binding antagonist is nivolumab.   16. The method of claim 15, wherein the PD-1 binding antagonist is pembrolizumab.   16. The method of claim 15, wherein the PD-1 binding antagonist is CT-011.   16. The method of claim 15, wherein the PD-1 binding antagonist is AMP-224.   15. The method of claim 14, wherein the PD-1 axis binding antagonist is a PDL1 binding antagonist.   26. The method of claim 25, wherein the PDL1 binding antagonist inhibits PDL1 binding to PD-1.   26. The method of claim 25, wherein the PDL1 binding antagonist inhibits PDL1 binding to B7-1.   26. The method of claim 25, wherein the PDL1 binding antagonist inhibits binding of PDL1 to both PD-1 and B7-1.   29. The method of any one of claims 25 to 28, wherein the PDL1 binding antagonist is an anti-PDL1 antibody.   30. The method of claim 29, wherein the anti-PDL1 antibody is a monoclonal antibody. 30. The method of claim 29, wherein the anti-PDL1 antibody is an antibody fragment selected from the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab ′) ₂ fragments.   30. The method of claim 29, wherein the anti-PDL1 antibody is a humanized antibody or a human antibody.   The PDL1 binding antagonist is YW243.55. 26. The method of claim 25, selected from the group consisting of S70, MPDL3280A, MDX-1105, and MEDI4736.   The antibody comprises a heavy chain comprising the HVR-H1 sequence GFTFSDSWHIH (SEQ ID NO: 1), HVR-H2 sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and the HVR-H3 sequence RHWPGGFDY (SEQ ID NO: 3), and the HVR-L1 sequence RASQDVSTAVA (sequence) 26. The method of claim 25, comprising: No. 4), a light chain comprising the HVR-L2 sequence SASFLYS (SEQ ID NO: 5), and the HVR-L3 sequence QQYLYHPAT (SEQ ID NO: 6).   Wherein the antibody comprises the amino acid sequence IbuikyuerubuiiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesudiesudaburyuaieichidaburyubuiarukyueiPijikeijieruidaburyubuieidaburyuaiesuPiwaijijiesutiwaiwaieidiesubuikeijiaruefutiaiesueiditiesukeienutieiwaierukyuemuenuesueruarueiiditieibuiYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK a heavy chain variable region (SEQ ID NO: 8), amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKL IY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR containing a light chain variable region (SEQ ID NO: 9) The method of claim 29.   15. The method of claim 14, wherein the PD-1 axis binding antagonist is a PDL2 binding antagonist.   38. The method of claim 36, wherein the PDL2 binding antagonist is an antibody.   40. The method of claim 36, wherein the PDL2 binding antagonist is an immunoadhesin.   38. The method of any one of claims 20, 29-35, and 37, wherein the antibody is human IgGl with an Asn to Ala substitution at position 297 according to EU numbering.   40. The method of any one of claims 1-39, wherein the OX40 binding agonist is selected from the group consisting of an OX40 agonist antibody, an OX40L agonist fragment, an OX40 oligomer receptor, and an OX40 immunoadhesin.   41. The method of any one of claims 1-40, wherein the OX40 binding agonist is an OX40 agonist antibody that binds to human OX40.   42. The method of claim 41, wherein the OX40 agonist antibody is MEDI6469, MEDI0562, or MEDI6383.   42. The method of claim 41, wherein the OX40 agonist antibody is a full length human IgGl antibody.   41. The method of any one of claims 1-40, wherein the OX40 binding agonist is a trimeric OX40L-Fc protein.   41. The method of any one of claims 1 to 40, wherein the OX40 binding agonist is an OX40L agonist fragment comprising one or more extracellular domains of OX40L.   The cancer is breast cancer, lung cancer, ovarian cancer, stomach cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, Or the method according to any one of claims 1 to 45, which is hepatocellular carcinoma.   47. The method of any one of claims 1-46, wherein the treatment results in a sustained response in the individual after cessation of the treatment.   48. The OX40 binding agonist according to any one of claims 1 to 47, wherein the OX40 binding agonist is administered before the PD-1 axis binding antagonist, simultaneously with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist. The method according to item.   49. The method of any one of claims 1-48, wherein the individual is a human.   A method for enhancing immune function in an individual having cancer, said method comprising administering an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist, wherein said individual has been diagnosed with cancer, and said individual The method, wherein the cancer cells in the tumor sample of the cancer derived from do not express PD-L1.   51. The method of claim 50, wherein the PD-L1 biomarker is not present in the sample when included in 0% of the sample.   52. The method of claim 51, wherein the PD-L1 biomarker is determined by protein expression measured by an immunohistochemistry (IHC) method.   A method for enhancing immune function in an individual having cancer, said method comprising administering an effective amount of a PD-1 axis binding antagonist and an OX40 binding agonist, wherein said individual has been diagnosed with cancer, and said individual The method, wherein the cancer cells in the cancer sample of origin express PD-L1.   54. The method of claim 53, wherein the PD-L1 biomarker is present in the sample when contained in greater than 0% of the sample.   55. The method of claim 53 or claim 54, wherein the PD-L1 biomarker is detected in 0% to 1% of the sample.   55. The method of claim 53 or claim 54, wherein the PD-L1 biomarker is detected in 0% to 5% of the sample.   55. The method of claim 54, wherein the PD-L1 biomarker is detected in the sample by protein expression as determined by immunohistochemistry (IHC) methods.   The PD-L1 biomarker is detected using an anti-PDL1 antibody, and the PD-L1 biomarker is detected as a weak staining intensity by IHC, a moderate staining intensity by IHC, or a strong staining intensity by IHC 58. The method of claim 57, wherein:   The PD-L1 biomarker is detected using an anti-PD-L1 antibody, and the PD-L1 biomarker is detected as moderate staining intensity by IHC or strong staining intensity by IHC. 58. The method according to 57.   60. The method of any one of claims 57 to 59, wherein the sample has an IHC score of IHC0 or IHC1.   61. The method of any one of claims 50-60, wherein the individual has a cancer that is resistant to a PD-1 axis binding antagonist.   62. The method of any one of claims 50-61, wherein the individual is refractory to a PD-1 axis binding antagonist.   The CD8 T cells in the individual have enhanced priming, activation, proliferation, and / or cytolytic activity as compared to prior to the administration of the PD-1 axis binding antagonist and the OX40 binding agonist. The method according to any one of 50 to 62.   63. The method of any one of claims 50-62, wherein the number of CD8 T cells is increased compared to before administration of the combination.   65. The method of claim 64, wherein the CD8 T cell is an antigen-specific CD8 T cell.   63. A method according to any one of claims 50 to 62, wherein Treg function is inhibited compared to before administration of the combination.   63. The method according to any one of claims 50 to 62, wherein T cell depletion is reduced compared to prior to the administration of the combination.   63. The method of any one of claims 50-62, wherein the number of Tregs is reduced compared to before administration of the combination.   63. The method of any one of claims 50-62, wherein plasma interferon gamma is increased compared to before administration of the combination.   63. The method of any one of claims 50-62, wherein memory effector T cell levels are increased compared to prior to the administration of the combination.   71. The method of claim 70, wherein the increase in the memory effector T cell level is detected in peripheral blood.   72. The method of claim 71, wherein the detection of increased memory effector T cell levels is by detection of CXCR3-expressing cells.   73. The method of any one of claims 50-72, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PDL1 binding antagonist, and a PDL2 binding antagonist.   74. The method of claim 73, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.   75. The method of claim 74, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner.   75. The method of claim 74, wherein the PD-1 binding antagonist inhibits PD-1 binding to PDL1.   75. The method of claim 74, wherein the PD-1 binding antagonist inhibits PD-1 binding to PDL2.   75. The method of claim 74, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PDL1 and PDL2.   79. The method according to any one of claims 74 to 78, wherein the PD-1 binding antagonist is an antibody.   75. The method of claim 74, wherein the PD-1 binding antagonist is nivolumab.   75. The method of claim 74, wherein the PD-1 binding antagonist is pembrolizumab.   75. The method of claim 74, wherein the PD-1 binding antagonist is CT-011.   75. The method of claim 74, wherein the PD-1 binding antagonist is AMP-224.   74. The method of claim 73, wherein the PD-1 axis binding antagonist is a PDL1 binding antagonist.   85. The method of claim 84, wherein the PDL1 binding antagonist inhibits PDL1 binding to PD-1.   85. The method of claim 84, wherein the PDL1 binding antagonist inhibits PDL1 binding to B7-1.   85. The method of claim 84, wherein the PDL1 binding antagonist inhibits binding of PDL1 to both PD-1 and B7-1.   88. The method of any one of claims 84 to 87, wherein the PDL1 binding antagonist is an anti-PDL1 antibody.   90. The method of claim 88, wherein the anti-PDL1 antibody is a monoclonal antibody. 90. The method of claim 88, wherein the anti-PDL1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab ') ₂ fragments.   90. The method of claim 88, wherein the anti-PDL1 antibody is a humanized antibody or a human antibody.   The PDL1 binding antagonist is YW243.55. 85. The method of claim 84, selected from the group consisting of S70, MPDL3280A, MDX-1105, and MEDI4736.   The anti-PDL1 antibody comprises a heavy chain comprising the HVR-H1 sequence GFTFSDSWHIH (SEQ ID NO: 1), the HVR-H2 sequence AWISPYGGSTYADSVKG (SEQ ID NO: 2), and the HVR-H3 sequence RHWPGGFDY (SEQ ID NO: 3), and the HVR-L1 sequence RASQDVSTAVA 90. The method of claim 88, comprising: (SEQ ID NO: 4), a HVR-L2 sequence SASFLYS (SEQ ID NO: 5), and a light chain comprising the HVR-L3 sequence QQYLYHPAT (SEQ ID NO: 6).   Wherein the anti-PDL1 antibody, the amino acid sequence IbuikyuerubuiiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesudiesudaburyuaieichidaburyubuiarukyueiPijikeijieruidaburyubuieidaburyuaiesuPiwaijijiesutiwaiwaieidiesubuikeijiaruefutiaiesueiditiesukeienutieiwaierukyuemuenuesueruarueiiditieibuiYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK a heavy chain variable region (SEQ ID NO: 8), amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPG APKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR containing a light chain variable region (SEQ ID NO: 9) The method of claim 88.   95. The method of any one of claims 79, 88-91, 93, and 94, wherein the antibody is human IgGl with an Asn to Ala substitution at position 297 according to EU numbering.   74. The method of claim 73, wherein the PD-1 axis binding antagonist is a PDL2 binding antagonist.   99. The method of claim 96, wherein the PDL2 binding antagonist is an antibody.   99. The method of claim 96, wherein the PDL2 binding antagonist is an immunoadhesin.   99. The method of any one of claims 50-98, wherein the OX40 binding agonist is selected from the group consisting of an OX40 agonist antibody, an OX40L agonist fragment, an OX40 oligomer receptor, and an OX40 immunoadhesin.   100. The method of claim 99, wherein the OX40 binding agonist is an OX40 agonist antibody that binds to human OX40.   101. The method of claim 100, wherein the OX40 agonist antibody is MEDI6469, MEDI0562, or MEDI6383.   101. The method of claim 100, wherein the OX40 agonist antibody is a full-length IgG1 antibody.   99. The method of any one of claims 50-98, wherein the OX40 binding agonist is a trimeric OX40L-Fc protein.   99. The method of any one of claims 50 to 98, wherein the OX40 binding agonist is an OX40L agonist fragment comprising one or more extracellular domains of OX40L.   The cancer is breast cancer, lung cancer, ovarian cancer, stomach cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, 105. The method according to any one of claims 50 to 104, which is hepatocellular carcinoma.   106. The method of any one of claims 50-105, wherein the treatment results in a sustained response in the individual after cessation of the treatment.   107. The OX40 binding agonist according to any one of claims 50 to 106, wherein the OX40 binding agonist is administered before the PD-1 axis binding antagonist, simultaneously with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist. The method according to item.   108. The method according to any one of claims 50 to 107, wherein the individual is a human.   The PD-1 axis binding antagonist and / or the OX40 binding agonist is intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, by implantation, by inhalation, intrathecal, intraventricular, 109. The method according to any one of claims 1 to 108, wherein the method is administered intranasally.   110. The method of any one of claims 1-109, further comprising administering a chemotherapeutic agent to treat cancer or delay its progression.   Use of a human PD-1 axis binding antagonist in the manufacture of a medicament for treating or delaying the progression of cancer in an individual, said medicament comprising said human PD-1 axis binding antagonist and any pharmaceutical In the tumor sample of the cancer from the individual, wherein the treatment comprises administration of the medicament in combination with a composition comprising an OX40 binding agonist and any pharmaceutically acceptable carrier. Wherein the cells do not express PD-L1.   Use of a human PD-1 axis binding antagonist in the manufacture of a medicament for treating or delaying the progression of cancer in an individual, said medicament comprising said human PD-1 axis binding antagonist and any pharmaceutical In the tumor sample of the cancer from the individual, wherein the treatment comprises administration of the medicament in combination with a composition comprising an OX40 binding agonist and any pharmaceutically acceptable carrier. Wherein said cells express PD-L1.   Use of an OX40 binding agonist in the manufacture of a medicament for treating cancer or delaying its progression in an individual, said medicament comprising said OX40 binding agonist and any pharmaceutically acceptable carrier, The treatment comprises administration of the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and any pharmaceutically acceptable carrier, and the cells in the cancer tumor sample from the individual are PD -Use as described above, which does not express L1.   Use of an OX40 binding agonist in the manufacture of a medicament for treating or delaying progression of cancer in an individual, said medicament comprising said OX40 binding agonist and any pharmaceutically acceptable carrier, The treatment comprises administration of the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and any pharmaceutically acceptable carrier, and the cells in the cancer tumor sample from the individual are PD -Said use expressing Ll.   A composition comprising a human PD-1 axis binding antagonist and any pharmaceutically acceptable carrier for use in treating cancer or delaying its progression in an individual, said treatment comprising, 2 wherein the second composition comprises an OX40 binding agonist and an optional pharmaceutically acceptable carrier, and the cells in the tumor sample of the cancer from the individual are PD- The composition described above, which does not express L1.   A composition comprising a human PD-1 axis binding antagonist and any pharmaceutically acceptable carrier for use in treating cancer or delaying its progression in an individual, said treatment comprising: 2 in combination, wherein the second composition comprises an OX40 binding agonist and an optional pharmaceutically acceptable carrier, and the cells in the tumor sample of the cancer from the individual are PD- Said composition expressing L1.   A composition comprising an OX40 binding agonist and any pharmaceutically acceptable carrier for use in treating cancer or delaying its progression in an individual, wherein said treatment comprises a second composition of said composition Wherein the second composition comprises a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and the cells in the tumor sample of the cancer from the individual are PD- The composition described above, which does not express L1.   A composition comprising an OX40 binding agonist and any pharmaceutically acceptable carrier for use in treating cancer or delaying its progression in an individual, wherein said treatment comprises a second composition of said composition Wherein the second composition comprises a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and the cells in the tumor sample of the cancer from the individual are PD- Said composition expressing L1.   OX40 binding of a medicament comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier and a package insert for treating cancer or delaying its progression in an individual The package insert comprising instructions for administration in combination with a composition comprising an agonist and any pharmaceutically acceptable carrier, wherein cells in the tumor sample of the cancer from the individual are PD -The kit does not express L1.   OX40 binding of a medicament comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier and a package insert for treating cancer or delaying its progression in an individual The package insert comprising instructions for administration in combination with a composition comprising an agonist and any pharmaceutically acceptable carrier, wherein cells in the tumor sample of the cancer from the individual are PD -The kit expressing L1.   A kit comprising: a first medicament comprising a PD-1 axis binding antagonist and any pharmaceutically acceptable carrier; and a second medicament comprising an OX40 binding agonist and any pharmaceutically acceptable carrier. The kit further comprises a package insert comprising instructions for administering the first medicament and the second medicament for treating or delaying the progression of cancer in the individual, The kit, wherein the cells in the tumor sample of cancer do not express PD-L1.   A kit comprising: a first medicament comprising a PD-1 axis binding antagonist and any pharmaceutically acceptable carrier; and a second medicament comprising an OX40 binding agonist and any pharmaceutically acceptable carrier. The kit further comprises a package insert comprising instructions for administration of the first medicament and the second medicament for treating or delaying the progression of cancer in the individual, from the individual The kit, wherein cells in the tumor sample of cancer express PD-L1.   PD-1 axis binding of a medicament comprising an OX40 binding agonist and any pharmaceutically acceptable carrier and a package insert for treating cancer or delaying its progression in an individual The package insert comprising instructions for administration in combination with a composition comprising an antagonist and any pharmaceutically acceptable carrier, wherein cells in the tumor sample of the cancer from the individual are PD -The kit does not express L1.   PD-1 axis binding of a medicament comprising an OX40 binding agonist and any pharmaceutically acceptable carrier and a package insert for treating cancer or delaying its progression in an individual The package insert comprising instructions for administration in combination with a composition comprising an antagonist and any pharmaceutically acceptable carrier, wherein cells in the tumor sample of the cancer from the individual are PD -The kit expressing L1.

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